Secondary literature sources for the EMP24_GP25L domain

For each of the hand selected primary papers associated with the EMP24_GP25L domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus.

Zhang T, Wong SH, Tang BL, Xu Y, Peter F, Subramaniam VN, Hong W
J Cell Biol. 1997; 139: 1157-68

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15 degrees C. However, upon warming up from 15 to 37 degrees C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER-derived vesicles with the cis-Golgi membrane.

GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6.

Wong SH, Xu Y, Zhang T, Griffiths G, Lowe SL, Subramaniam VN, Seow KT, Hong W
Mol Biol Cell. 1999; 10: 119-34

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

An acidic sequence of a putative yeast Golgi membrane protein binds COPII and facilitates ER export.

Votsmeier C, Gallwitz D
EMBO J. 2001; 20: 6742-50

We previously identified Sys1p as a high copy number suppressor of Ypt6 GTPase-deficient yeast mutants that are defective in endosome-to-Golgi transport. Here, we show that Sys1p is an integral membrane protein that resides on a post-endoplasmic reticulum (ER) organelle(s). Affinity studies with detergent- solubilized yeast proteins showed that the C-terminal 53 amino acid tail of Sys1p binds effectively to the cytoplasmic Sec23p-Sec24p COPII subcomplex. This binding required a di-acidic Asp-Leu-Glu (DXE) motif, previously shown to mediate efficient ER export of the vesicular stomatitis virus glycoprotein in mammalian cells. In Sys1p, a Glu-Leu-Glu (EXE) sequence could not substitute for the (DXE) motif. Mutations of the (DXE) sequence resulted in ER retention of approximately 30% of the protein at steady state, whereas addition of the Sys1p tail to an ER-resident membrane protein led to an intracellular redistribution of the chimeric protein. Our study demonstrates for the first time that, in yeast, a di-acidic sequence motif can act as a sorting signal for cargo selection during the formation of transport vesicles at the ER by direct binding to COPII component(s).

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p.

VanRheenen SM, Cao X, Sapperstein SK, Chiang EC, Lupashin VV, Barlowe C, Waters MG
J Cell Biol. 1999; 147: 729-42

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

Formation of secretory vesicles in the biosynthetic pathway.

Urbe S, Tooze SA, Barr FA
Biochim Biophys Acta. 1997; 1358: 6-22

A conserved N-terminal arginine-motif in GOLPH3-family proteins mediates binding to coatomer.

Tu L, Chen L, Banfield DK
Traffic. 2012; 13: 1496-507

Vps74p, a member of the GOLPH3 protein family, binds directly to coatomer and the cytoplasmic tails of a subset of Golgi-resident glycosyltransferases to mediate their Golgi retention. We identify a cluster of arginine residues at the N-terminal end of GOLPH3 proteins that are necessary and sufficient to mediate coatomer binding. While loss of coatomer binding renders Vps74p non-functional for glycosyltransferase retention, the Golgi membrane-binding capabilities of the mutant protein are not significantly reduced. We establish that the oligomerization status and phosphatidylinositol-4-phosphate-binding properties of Vps74p largely account for the membrane-binding capacity of the protein and identify an Arf1p-Vps74p interaction as a potential contributing factor in Vps74p Golgi membrane association.

Endocytosis and signals for internalization.

Trowbridge IS
Curr Opin Cell Biol. 1991; 3: 634-41

The most important recent advance in the field of endocytosis has been the identification of the internalization signals of several constitutively recycling receptors. Common structural features and chemistry of internalization sequences have been defined, and an exposed tight turn has been implicated as the conformational recognition motif for endocytosis.

Identification of a mammalian Golgi Sec1p-like protein, mVps45.

Tellam JT, James DE, Stevens TH, Piper RC
J Biol Chem. 1997; 272: 6187-93

Our understanding of lysosomal biogenesis and general vesicular transport in animal cells has been greatly enhanced by studies of vacuolar biogenesis in yeast. Genetic screens have identified a number of proteins that play direct roles in the proper sorting of vacuolar hydrolases. These include t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and Sec1p-like proteins, which have recently been implicated as key regulators of vesicle fusion. In this study we have extended these observations in yeast and have isolated and characterized a novel member of the Sec1p-like family of proteins from mammalian cells, mVps45. mVps45 shares a high level of identity with the Saccharomyces cerevisiae Sec1p-like protein Vps45p that is believed to function with the t-SNARE Pep12p in the fusion of Golgi-derived transport vesicles with a prevacuolar compartment. We found that mVps45 is a ubiquitously expressed peripheral membrane protein that localized to perinuclear Golgi-like and trans-Golgi network compartments in Chinese hamster ovary cells. We found that mVps45 could bind specifically to yeast Pep12p and to the mammalian Pep12p-like protein, syntaxin 6, in vitro.

The cargo receptor p24A facilitates calcium sensing receptor maturation and stabilization in the early secretory pathway.

Stepanchick A, Breitwieser GE
Biochem Biophys Res Commun. 2010; 395: 136-40

The calcium sensing receptor (CaSR) is a Family 3/C G protein-coupled receptor with slow and partial targeting to the plasma membrane in both native and heterologous cells. We identified cargo receptor family member p24A in yeast two-hybrid screens with the CaSR carboxyl terminus. Interactions were confirmed by immunoprecipitation of either p24A or CaSR in transiently transfected HEK293 cells. Only the immaturely glycosylated form of CaSR interacts with p24A. Dissociation likely occurs in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) or cis-Golgi, since only the uncleaved form of a CaSR mutant sensitive to the trans-Golgi enzyme furin was co-immunoprecipitated with p24A. p24A and p24A(DeltaGOLD) significantly increased total and plasma membrane CaSR protein but p24A(FF/AA) did not. The CaSR carboxyl terminus distal to T868 is required for differential sensitivity to p24A and its mutants. Interaction with p24A therefore increases CaSR stability in the ER and enhances plasma membrane targeting. Neither wt Sar1p or the T39N mutant increased CaSR maturation or abundance while the H79G mutant increased abundance but prevented maturation of CaSR. These results suggest that p24A is the limiting factor in CaSR trafficking in the early secretory pathway, and that cycling between the ER and ERGIC protects CaSR from degradation.

The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae.

Springer S, Chen E, Duden R, Marzioch M, Rowley A, Hamamoto S, Merchant S, Schekman R
Proc Natl Acad Sci U S A. 2000; 97: 4034-9

To investigate the factors involved in the sorting of cargo proteins into COPII endoplasmic reticulum (ER) to Golgi apparatus transport vesicles, we have created a strain of S. cerevisiae (p24Delta8) that lacks all eight members of the p24 family of transmembrane proteins (Emp24p, Erv25p, and Erp1p to Erp6p). The p24 proteins have been implicated in COPI and COPII vesicle formation, cargo protein sorting, and regulation of vesicular transport in eukaryotic cells. We find that p24Delta8 cells grow identically to wild type and show delays of invertase and Gas1p ER-to-Golgi transport identical to those seen in a single Deltaemp24 deletion strain. Thus, p24 proteins do not have an essential function in the secretory pathway. Instead, they may serve as quality control factors to restrict the entry of proteins into COPII vesicles.

Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells.

Soupene E, Serikov V, Kuypers FA
J Lipid Res. 2008; 49: 1103-12

Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development.

Automatic generation of primary sequence patterns from sets of related protein sequences.

Smith RF, Smith TF
Proc Natl Acad Sci U S A. 1990; 87: 118-22

We have developed a computer algorithm that can extract the pattern of conserved primary sequence elements common to all members of a homologous protein family. The method involves clustering the pairwise similarity scores among a set of related sequences to generate a binary dendrogram (tree). The tree is then reduced in a stepwise manner by progressively replacing the node connecting the two most similar termini by one common pattern until only a single common "root" pattern remains. A pattern is generated at a node by (i) performing a local optimal alignment on the sequence/pattern pair connected by the node with the use of an extended dynamic programming algorithm and then (ii) constructing a single common pattern from this alignment with a nested hierarchy of amino acid classes to identify the minimal inclusive amino acid class covering each paired set of elements in the alignment. Gaps within an alignment are created and/or extended using a "pay once" gap penalty rule, and gapped positions are converted into gap characters that function as 0 or 1 amino acid of any type during subsequent alignment. This method has been used to generate a library of covering patterns for homologous families in the National Biomedical Research Foundation/Protein Identification Resource protein sequence data base. We show that a covering pattern can be more diagnostic for sequence family membership than any of the individual sequences used to construct the pattern.

Partial purification and characterization of early and late endosomes from yeast. Identification of four novel proteins.

Singer-Kruger B, Frank R, Crausaz F, Riezman H
J Biol Chem. 1993; 268: 14376-86

Previously (Singer, B., and Riezman, H. (1990) J. Cell Biol. 110, 1911-1922), we provided evidence for the existence of an endocytic intermediate(s) from the yeast Saccharomyces cerevisiae that is responsible for the transport of the pheromone alpha-factor from the plasma membrane to the vacuole. Here we show by kinetic analysis that the endocytic apparatus of yeast is composed of early and late endosomes, similar to what has been found in animal cells. We have developed a three-step isolation procedure to purify early and late endosomes, consisting of differential centrifugation, flotation on a Nycodenz density gradient, and sedimentation density gradient centrifugation on sucrose/D2O. Using internalized 35S-alpha-factor as a marker, the endosomal fractions were substantially enriched over other membranes, except for Golgi elements and a compartment containing binding protein. These contaminants could not be removed by other standard purification methods. We have analyzed the protein composition of our most pure early and late endosome fractions. By two-dimensional gel analysis we identified more than 20 proteins spots that are highly enriched in the early/late endosomal fractions. N-terminal protein sequencing resulted in the identification of four novel proteins.

Biogenesis of tubular ER-to-Golgi transport intermediates.

Simpson JC, Nilsson T, Pepperkok R
Mol Biol Cell. 2006; 17: 723-37

Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.

In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein.

Simon JP, Ivanov IE, Adesnik M, Sabatini DD
Methods. 2000; 20: 437-54

We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus.

Schweizer A, Fransen JA, Matter K, Kreis TE, Ginsel L, Hauri HP
Eur J Cell Biol. 1990; 53: 185-96

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.

Sterol transport in yeast and the oxysterol binding protein homologue (OSH) family.

Schulz TA, Prinz WA
Biochim Biophys Acta. 2007; 1771: 769-80

Sterols such as cholesterol are a significant component of eukaryotic cellular membranes, and their unique physical properties influence a wide variety of membrane processes. It is known that the concentration of sterol within the membrane varies widely between organelles, and that the cell actively maintains this distribution through various transport processes. Vesicular pathways such as secretion or endocytosis may account for this traffic, but increasing evidence highlights the importance of nonvesicular routes as well. The structure of an oxysterol-binding protein homologue (OSH) in yeast (Osh4p/Kes1p) has recently been solved, identifying it as a sterol binding protein, and there is evidence consistent with the role of a cytoplasmic, nonvesicular sterol transporter. Yeast have seven such proteins, which appear to have distinct but overlapping functions with regard to maintaining intracellular sterol distribution and homeostasis. Control of sterol distribution can have far-reaching effects on membrane-related functions, and Osh proteins have been implicated in a variety of processes such as secretory vesicle budding from the Golgi and establishment of cell polarity. This review summarizes the current body of knowledge regarding this family and its potential functions, placing it in the context of known and hypothesized pathways of sterol transport in yeast.

Rer1p, a retrieval receptor for endoplasmic reticulum membrane proteins, is dynamically localized to the Golgi apparatus by coatomer.

Sato K, Sato M, Nakano A
J Cell Biol. 2001; 152: 935-44

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

Membrane protein retrieval from the Golgi apparatus to the endoplasmic reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Sato K, Nishikawa S, Nakano A
Mol Biol Cell. 1995; 6: 1459-77

Yeast Sec12p, a type II transmembrane glycoprotein, is required for formation of transport vesicles from the endoplasmic reticulum (ER). Biochemical and morphological analyses have suggested that Sec12p is localized to the ER by two mechanisms: static retention in the ER and dynamic retrieval from the early region of the Golgi apparatus. The rer1 mutant we isolated in a previous study mislocalizes the authentic Sec12p to the later compartments of the Golgi. To understand the role of RER1 on Sec12p localization, we cloned the gene and determined its reading frame. RER1 encodes a hydrophobic protein of 188 amino acid residues containing four putative membrane spanning domains. The rer1 null mutant is viable. Even in the rer1 disrupted cells, immunofluorescence of Sec12p stains the ER, implying that the retention system is still operating in the mutant. To determine the subcellular localization of Rer1p, an epitope derived from the influenza hemagglutinin was added to the C-terminus of Rer1p and the cells expressing this tagged but functional protein were observed by immunofluorescence microscopy. The anti-HA monoclonal antibody stains the cells in a punctate pattern that is typical for Golgi proteins and clearly distinct from the ER staining. This punctate staining was in fact exaggerated in the sec7 mutant that accumulates the Golgi membranes at the restrictive temperature. Furthermore, double staining of Rer1p and Ypt1p, a GTPase that is known to reside in the Golgi apparatus, showed good colocalization. Subcellular fractionation experiments indicated that the fractionation pattern of Rer1p was similar to that of an early Golgi protein, Och1p. From these results, we suggest that Rer1p functions in the Golgi membrane to return Sec12p that has escaped from the static retention system of the ER.

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

Satoh T, Sato K, Kanoh A, Yamashita K, Yamada Y, Igarashi N, Kato R, Nakano A, Wakatsuki S
J Biol Chem. 2006; 281: 10410-9

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum.

Roy L, Bergeron JJ, Lavoie C, Hendriks R, Gushue J, Fazel A, Pelletier A, Morre DJ, Subramaniam VN, Hong W, Paiement J
Mol Biol Cell. 2000; 11: 2529-42

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors.

Rotter J, Kuiper RP, Bouw G, Martens GJ
J Cell Sci. 2002; 115: 1049-58

Members of the p24 family of type I transmembrane proteins are highly abundant in transport vesicles and are thought to be involved in selective protein transport between the endoplasmic reticulum and the Golgi complex. The p24 proteins have been grouped into four subfamilies (alpha, beta, gamma, and delta) and appear to assemble into tetrameric complexes that contain only one representative from each subfamily. Here we molecularly dissected the p24 family in a single cell type, namely in the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis. The biosynthetic activity of these cells for production of their major cargo protein proopiomelanocortin (POMC) can be physiologically manipulated via the process of background adaptation (similar30-fold induction, with highly active cells in black toads and virtually inactive cells in white animals). Extensive cDNA library screening revealed the identity of six p24 proteins expressed in the Xenopus melanotrope cells, namely one member of the p24alpha (alpha(3)), one of the p24beta (beta(1)), two of the p24gamma (gamma(2), gamma(3)) and two of the p24delta (delta(1), delta(2)) subfamily. Two other Xenopus p24 proteins, Xp24alpha(2) and -gamma(1), were not expressed in the melanotrope cells, pointing to cell-type specific p24 expression. Of the six melanotrope p24 proteins, the expression of four (Xp24alpha(3), -beta(1), -gamma(3) and -delta(2)) was 20- to 30-fold induced in active versus inactive melanotropes, whereas that of the other two members (Xp24gamma(2) and -delta(1)) had not or only slightly increased. The four proteins were induced only in the intermediate melanotrope cells and not in the anterior pituitary cells, and displayed similar overall tissue distributions that differed from those of Xp24gamma(1), -gamma(2) and -delta(1). Together, our results reveal that p24 expression can be cell-type specific and selectively induced, and suggest that in Xenopus melanotrope cells an alpha(3)/beta(1)/gamma(3)/delta(2) p24 complex is involved in POMC transport through the early stages of the secretory pathway.

Involvement of the transmembrane protein p23 in biosynthetic protein transport.

Rojo M, Pepperkok R, Emery G, Kellner R, Stang E, Parton RG, Gruenberg J
J Cell Biol. 1997; 139: 1119-35

Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/micron2 membrane surface area, or approximately 30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment. Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport.

The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex.

Rohde HM, Cheong FY, Konrad G, Paiha K, Mayinger P, Boehmelt G
J Biol Chem. 2003; 278: 52689-99

The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus. Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations. At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity. Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific. Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described. Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1. Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi. In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif. These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.

The exomer cargo adaptor features a flexible hinge domain.

Richardson BC, Fromme JC
Structure. 2013; 21: 486-92

Exomer is a cargo adaptor that mediates the sorting of specific plasma membrane proteins into vesicles at the trans-Golgi network. Cargo adaptors must bind to multiple partners, including their cargo, regulatory proteins, and the membrane surface. During biogenesis of a vesicle, the membrane makes a transition from a relatively flat surface to one of high curvature, requiring cargo adaptors to somehow maintain protein-protein and protein-membrane interactions on a changing membrane environment. Here, we present the crystal structure of a tetrameric Chs5/Bch1 exomer complex and use small-angle X-ray scattering to demonstrate its flexibility in solution. The structural data suggest that the complex flexes primarily around the dimeric N-terminal domain of the Chs5 subunits, which adopts a noncanonical beta sandwich fold. We propose that this flexible hinge domain enables exomer to maintain interactions in the context of a dynamic membrane environment.

Nitric oxide-induced endoplasmic reticulum stress activates the expression of cargo receptor proteins and alters the glycoprotein transport to the Golgi complex.

Renna M, Faraonio R, Bonatti S, De Stefano D, Carnuccio R, Tajana G, Remondelli P
Int J Biochem Cell Biol. 2006; 38: 2040-8

The endoplasmic reticulum Golgi intermediate compartment 53 protein recycles continuously between the endoplasmic reticulum and the Golgi complex and ensures the anterograde transport of specific glycoproteins with the assistance of the Multiple Clotting Factor Deficiency adaptor protein. Therefore, to analyze the effect of the endoplasmic reticulum stress on the secretory pathway beyond the endoplasmic reticulum, we analyzed the expression of both proteins in J774 macrophages incubated with the nitric oxide donor DETA NONOate or with thapsigargin. Both proteins accumulated progressively, by a transcriptional mechanism, in response to these inducers. Nitric oxide also induced a higher level of calreticulin and glucose regulated 78 protein, two endoplasmic reticulum proteins controlled by the unfolded protein response. Interestingly, nitric oxide induced the processing of the activating transcription factor 6alpha of the unfolded protein response, while thapsigargin also induced the activation of the transcription factor X-box Binding Protein 1. In addition, we showed that the accumulation of both transporters occurred simultaneously with the activation of endoplasmic reticulum-stress-dependent apoptosis, suggesting that these proteins may participate in the events that will eventually decide the fate of the cell. Induction of endoplasmic reticulum stress affected the rate of anterograde transport of a reporter glycoprotein, indicating that the endoplasmic reticulum to Golgi transport is remarkably impaired. Our results indicate that increased levels of cargo receptor proteins might have a function either in the quality control of protein folding in the endoplasmic reticulum or in the homeostasis of the intermediate compartment and Golgi complex during cell stress.

Functional reconstitution of COPI coat assembly and disassembly using chemically defined components.

Reinhard C, Schweikert M, Wieland FT, Nickel W
Proc Natl Acad Sci U S A. 2003; 100: 8253-7

Coat protein I (COPI)-coated transport vesicles mediate protein and lipid transport in the early secretory pathway. The basic machinery required for the formation of these transport intermediates has been elucidated based on the reconstitution of COPI-coated vesicle formation from chemically defined liposomes. In this experimental system, the coat components coatomer and GTP-bound ADP-ribosylation factor (ARF), as well as p23 as a membrane-bound receptor for COPI coat proteins, were shown to be both necessary and sufficient to promote COPI-coated vesicle formation. Based on biochemical and ultrastructural analyses, we now demonstrate that the catalytic domain of ARF-GTPase-activating protein (GAP) alone is sufficient to initiate uncoating of liposome-derived COPI-coated vesicles. By contrast, ARF-GAP activity is not required for COPI coat assembly and, therefore, does not seem to represent an essential coat component of COPI vesicles as suggested recently [Yang, J. S., Lee, S. Y., Gao, M., Bourgoin, S., Randazzo, P. A., et al. (2002) J. Cell Biol. 159, 69-78]. Thus, a complete round of COPI coat assembly and disassembly has been reconstituted with purified components defining the core machinery of COPI vesicle biogenesis.

ER-to-Golgi transport visualized in living cells.

Presley JF, Cole NB, Schroer TA, Hirschberg K, Zaal KJ, Lippincott-Schwartz J
Nature. 1997; 389: 81-5

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. We have now visualized ER-to-Golgi transport using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP became concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocated inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurred during their translocation to the Golgi complex and they frequently stretched into tubular shapes. Together, our results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport.

Vear, a novel Golgi-associated protein with VHS and gamma-adaptin "ear" domains.

Poussu A, Lohi O, Lehto VP
J Biol Chem. 2000; 275: 7176-83

The molecular basis of the selectivity and the details of the vesicle formation in endocytic and secretory pathways are still poorly known and most probably involve as yet unidentified components. Here we describe the cloning, expression, and tissue and cell distribution of a novel protein of 67 kDa (called Vear) that bears homology to several endocytosis-associated proteins in that it has a VHS domain in its N terminus. It is also similar to gamma-adaptin, the heavy subunit of AP-1, in having in its C terminus a typical "ear" domain. In immunofluorescence microscopy, Vear was seen in the Golgi complex as judged by a typical distribution pattern, a distinct colocalization with the Golgi marker gamma-adaptin, and a sensitivity to treatment of cells with brefeldin A. In cell fractionation, Vear partitioned with the post-nuclear membrane fraction. In transfection experiments, hemagglutinin-tagged full-length Vear and truncated Vear lacking the VHS domain assembled on and caused compaction of the Golgi complex. Golgi association without compaction was seen with the ear domain of Vear, whereas the VHS domain alone showed a diffuse membrane- and vesicle-associated distribution. The Golgi association and the bipartite structure along with the differential targeting of its domains suggest that Vear is involved in heterotypic vesicle/suborganelle interactions associated with the Golgi complex. Tissue-specific function of Vear is suggested by its high level of expression in kidney, muscle, and heart.

A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.

Port F, Hausmann G, Basler K
EMBO Rep. 2011; 12: 1144-52

Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.

Mammalian homologues of yeast vacuolar protein sorting (vps) genes implicated in Golgi-to-lysosome trafficking.

Pevsner J, Hsu SC, Hyde PS, Scheller RH
Gene. 1996; 183: 7-14

Sec1p, Vps33p, Vps45p and Sly1p constitute a family of proteins implicated in vesicle trafficking at distinct stages of the yeast secretory pathway. Several mammalian homologues of Sec1p have been described, including n-sec1 which has been implicated in the regulation of synaptic vesicle docking at the nerve terminal. We have characterized cDNA clones encoding three additional mammalian homologues belonging to this family: r-vps33a and r-vps33b from rat, which are 30 and 26% identical to yeast Vps33p, respectively, and h-vps45 from human which is 38% identical to yeast Vps45p at the amino acid (aa) level. Phylogenetic analysis of 16 Sec1p-related proteins from several species is consistent with the hypothesis that the evolution of this gene family parallels the specialization of vesicle trafficking to distinct intracellular compartments. By Northern analysis, each of these genes is expressed in all tissues examined (brain, spleen, lung, liver, skeletal muscle, kidney, testis). While n-sec1 binds syntaxin 1a, 2, and 3, r-vps33a, r-vps33b and h-vps45 do not bind any of the known syntaxins. We propose that the three proteins bind as yet unidentified syntaxin homologues involved in vesicle trafficking between the Golgi apparatus, prelysosomal compartment(s), and the lysosome.

Orphan macrodomain protein (human C6orf130) is an O-acyl-ADP-ribose deacylase: solution structure and catalytic properties.

Peterson FC, Chen D, Lytle BL, Rossi MN, Ahel I, Denu JM, Volkman BF
J Biol Chem. 2011; 286: 35955-65

Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD(+)-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD(+)-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the beta(5)-alpha(4) loop that covers the bound ADPr, suggesting that the beta(5)-alpha(4) loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues.

Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export.

Pathre P, Shome K, Blumental-Perry A, Bielli A, Haney CJ, Alber S, Watkins SC, Romero G, Aridor M
EMBO J. 2003; 22: 4059-69

The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.

Association of folding intermediates of glycoproteins with calnexin during protein maturation.

Ou WJ, Cameron PH, Thomas DY, Bergeron JJ
Nature. 1993; 364: 771-6

Calnexin, an endoplasmic reticulum transmembrane protein, represents a new type of molecular chaperone that selectively associates in a transient fashion with newly synthesized monomeric glycoproteins in HepG2 cells. Calnexin only recognizes glycoproteins when they are incompletely folded. Dissociation of glycoproteins from calnexin occurs at different rates and is related to the time taken for their folding, which may then initiate their differential transport rates from the endoplasmic reticulum.

Bidirectional transport by distinct populations of COPI-coated vesicles.

Orci L, Stamnes M, Ravazzola M, Amherdt M, Perrelet A, Sollner TH, Rothman JE
Cell. 1997; 90: 335-49

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

Beta-COP localizes mainly to the cis-Golgi side in exocrine pancreas.

Oprins A, Duden R, Kreis TE, Geuze HJ, Slot JW
J Cell Biol. 1993; 121: 49-59

We examined the distribution of the non-clathrin-coated vesicle-associated coat protein beta-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for beta-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of approximately 50 nm, showing a characteristic approximately 10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of approximately 18 nm. Of the total beta-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans-side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side beta-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while beta-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated beta-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of beta-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of beta-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of beta-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for beta-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of beta-COP. Our data suggest that beta-COP plays a role in membrane transport at the cis-side of the Golgi complex.

Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.

Nielsen H, Engelbrecht J, Brunak S, von Heijne G
Protein Eng. 1997; 10: 1-6

We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW server.

p23, a major COPI-vesicle membrane protein, constitutively cycles through the early secretory pathway.

Nickel W, Sohn K, Bunning C, Wieland FT
Proc Natl Acad Sci U S A. 1997; 94: 11393-8

A novel type I transmembrane protein of COPI-coated vesicles, p23, has been demonstrated to be localized mainly to the Golgi complex. This protein and p24, another member of the p24 family, have been shown to bind coatomer via their short cytoplasmic tails. Here we demonstrate that p23 continuously cycles through the early secretory pathway. The cytoplasmic tail of p23 is shown to act as a functional retrieval signal as it confers endoplasmic reticulum (ER) residence to a CD8-p23 fusion protein. This ER localization is, at least in part, a result of retrieval from post-ER compartments because CD8-p23 fusion proteins receive post-ER modifications. In contrast, the cytoplasmic tail of p24 has been shown not to retrieve a CD8-p24 fusion protein. The coatomer binding motifs FF and KK in the cytoplasmic tail of p23 are reported to influence the steady-state localization of the CD8-p23 fusion protein within the ER-Golgi recycling pathway. It appears that the steady-state Golgi localization of endogenous p23 is maintained by its lumenal domain, as a fusion protein with the lumenal domain of CD8, and the membrane span as well as the cytoplasmic tail of p23 is no longer detected in the Golgi.

The membrane transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for its localization and function.

Nelson DS, Alvarez C, Gao YS, Garcia-Mata R, Fialkowski E, Sztul E
J Cell Biol. 1998; 143: 319-31

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al.; 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15 degreesC treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.

Characterization of a cis-Golgi matrix protein, GM130.

Nakamura N, Rabouille C, Watson R, Nilsson T, Hui N, Slusarewicz P, Kreis TE, Warren G
J Cell Biol. 1995; 131: 1715-26

Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER-Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.

Emerging new roles of GM130, a cis-Golgi matrix protein, in higher order cell functions.

Nakamura N
J Pharmacol Sci. 2010; 112: 255-64

GM130 is a peripheral membrane protein strongly attached to the Golgi membrane and is isolated from the detergent and salt resistant Golgi matrix. GM130 is rich in coiled-coil structures and predicted to take a rod-like shape. Together with p115, giantin, and GRASP65, GM130 facilitates vesicle fusion to the Golgi membrane as a vesicle "tethering factor". GM130 is also involved in the maintenance of the Golgi structure and plays a major role in the disassembly and reassembly of the Golgi apparatus during mitosis. Emerging evidence suggests that GM130 is involved in the control of glycosylation, cell cycle progression, and higher order cell functions such as cell polarization and directed cell migration. This creates the potential for novel Golgi-targeted drugs and treatments for various diseases including glycosylation defects, immune diseases, and cancer.

Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway.

Montesinos JC, Langhans M, Sturm S, Hillmer S, Aniento F, Robinson DG, Marcote MJ
J Exp Bot. 2013; 64: 3147-67

p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24alpha, p24beta, p24gamma, and p24delta. In contrast to animals and fungi, plants contain only members of the p24beta and p24delta subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)-p24delta5 (p24delta1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP-p24delta9 (p24delta2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)-p24beta3 mainly localizes to the Golgi apparatus (as p24beta2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24delta9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24beta3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the beta and delta subfamilies) which are important for their stability and their coupled trafficking at the ER-Golgi interface. Evidence is also provided for a role for p24delta5 in retrograde Golgi-ER transport of the KDEL-receptor ERD2.

A putative modular domain present in diverse signaling proteins.

Mayer BJ, Ren R, Clark KL, Baltimore D
Cell. 1993; 73: 629-30

Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4.

Mari M, Macia E, Le Marchand-Brustel Y, Cormont M
J Biol Chem. 2001; 276: 42501-8

Rabip4 is a Rab4 effector, which possesses a RUN domain, two coiled-coil domains, and a FYVE finger. It is associated with the early endosomes and leads, in concert with Rab4, to the enlargement of endosomes, resulting in the fusion of sorting and recycling endosomes. Our goal was to characterize the role of these various domains in Rabip4 subcellular localization and their function in Chinese hamster ovary cells. Although the FYVE finger domain specifically bound phosphatidylinositol 3-phosphate and was necessary for the function of Rabip4, it was not sufficient for the protein association with membranes. Indeed a protein containing the FYVE finger and the Rab4-binding site was cytosolic, whereas the total protein was mostly associated to the membrane fraction, whether or not cells were pretreated with wortmannin. By contrast, a construct corresponding to the N-terminal end, Rabip4-(1-212), and containing the RUN domain was membrane-associated. The complete protein partitioned between the Triton X-100-insoluble and -soluble fractions and a wortmannin treatment increased the amount of the protein in the Triton X-100 fraction. Rabip4-(1-212) was totally Triton X-100-insoluble, and confocal microscopic examination showed that it labeled not only the endosomes, positive for Rabip4, but also a filamentous network with a honeycomb appearance. The Triton X-100-insoluble fraction that contains Rabip4 did not correspond to the caveolin or glycosylphosphatidylinositol-enriched lipid rafts. Rabip4 did not appear directly linked to actin but seemed associated to the actin network. We propose that the subcellular localization of the protein is primarily driven by the RUN domain to endosomal microdomains characterized by Triton X-100 insolubility and that the FYVE domain and the Rab4-binding domain then allow for the recruitment of the protein to lipophilic microdomains enriched in phosphatidylinositol 3-phosphate.

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network.

Luke MR, Kjer-Nielsen L, Brown DL, Stow JL, Gleeson PA
J Biol Chem. 2003; 278: 4216-26

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.

Loh E, Hong W
J Biol Chem. 2002; 277: 21955-61

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.

A human homologue of the yeast HDEL receptor.

Lewis MJ, Pelham HR
Nature. 1990; 348: 162-3

Retention of resident proteins in the lumen of the endoplasmic reticulum is achieved in both yeast and animal cells by their continual retrieval from the cis-Golgi, or a pre-Golgi compartment. Sorting of these proteins is dependent on a C-terminal tetrapeptide signal, usually Lys-Asp-Glu-Leu (KDEL in the single letter code) in animal cells, His-Asp-Glu-Leu (HDEL) in Saccharomyces cerevisiae. There is evidence that the ERD2 gene encodes the sorting receptor that recognizes HDEL in yeast; its product is an integral membrane protein of relative molecular mass 26,000 (26K) that is not glycosylated. In contrast, Vaux et al. suggest that the mammalian KDEL receptor is a 72K glycoprotein that they detected using an anti-idiotypic antibody approach. If this were so, it would indicate a surprising divergence of the retrieval machinery between yeast and animal cells. We report here that human cells express a protein similar in sequence, size and properties to the ERD2 product, and propose that this protein is the human KDEL receptor.

Tyrosine phosphorylation of p97 regulates transitional endoplasmic reticulum assembly in vitro.

Lavoie C, Chevet E, Roy L, Tonks NK, Fazel A, Posner BI, Paiement J, Bergeron JJ
Proc Natl Acad Sci U S A. 2000; 97: 13637-42

The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M, Nilsson T
J Cell Biol. 2001; 155: 1199-212

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Sec24p and Iss1p function interchangeably in transport vesicle formation from the endoplasmic reticulum in Saccharomyces cerevisiae.

Kurihara T, Hamamoto S, Gimeno RE, Kaiser CA, Schekman R, Yoshihisa T
Mol Biol Cell. 2000; 11: 983-98

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression of ISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Delta mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations in SEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His(6) at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (alpha-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.

Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell.

Kuiper RP, Waterham HR, Rotter J, Bouw G, Martens GJ
Mol Biol Cell. 2000; 11: 131-40

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24alpha, -beta, -gamma, and -delta. We previously identified a member of the p24delta subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis ( approximately 30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24delta member (Xp24delta(2)) and on the identification and characterization of a second member (Xp24delta(1)) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24delta(2). The two p24delta members are ubiquitously expressed, but Xp24delta(2) is neuroendocrine enriched. During black background adaptation, the amount of the Xp24delta(2) protein in the intermediate pituitary was increased approximately 25 times, whereas Xp24delta(1) protein expression was increased only 2.5 times. Furthermore, the level of Xp24delta(2) mRNA was approximately 5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24delta(1) mRNA expression was not induced. Therefore, the expression of Xp24delta(2) specifically correlates with the expression of POMC. Together, our findings suggest that p24delta proteins have a role in selective protein transport in the secretory pathway.

dGRASP localization and function in the early exocytic pathway in Drosophila S2 cells.

Kondylis V, Spoorendonk KM, Rabouille C
Mol Biol Cell. 2005; 16: 4061-72

The de novo model for Golgi stack biogenesis predicts that membrane exiting the ER at transitional ER (tER) sites contains and recruits all the necessary molecules to form a Golgi stack, including the Golgi matrix proteins, p115, GM130, and GRASP65/55. These proteins leave the tER sites faster than Golgi transmembrane resident enzymes, suggesting that they act as a template nucleating the formation of the Golgi apparatus. However, the localization of the Golgi matrix proteins at tER sites is only shown under conditions where exit from the ER is blocked. Here, we show in Drosophila S2 cells, that dGRASP, the single Drosophila homologue of GRASP65/55, localizes both to the Golgi membranes and the tER sites at steady state and that the myristoylation of glycine 2 is essential for the localization to both compartments. Its depletion for 96 h by RNAi gave an effect on the architecture of the Golgi stacks in 30% of the cells, but a double depletion of dGRASP and dGM130 led to the quantitative conversion of Golgi stacks into clusters of vesicles and tubules, often featuring single cisternae. This disruption of Golgi architecture was not accompanied by the disorganization of tER sites or the inhibition of anterograde transport. This shows that, at least in Drosophila, the structural integrity of the Golgi stacks is not required for efficient transport. Overall, dGRASP exhibits a dynamic association to the membrane of the early exocytic pathway and is involved in Golgi stack architecture.

Longin-like folds identified in CHiPS and DUF254 proteins: vesicle trafficking complexes conserved in eukaryotic evolution.

Kinch LN, Grishin NV
Protein Sci. 2006; 15: 2669-74

Eukaryotic protein trafficking pathways require specific transfer of cargo vesicles to different target organelles. A number of vesicle trafficking and membrane fusion components participate in this process, including various tethering factor complexes that interact with small GTPases prior to SNARE-mediated vesicle fusion. In Saccharomyces cerevisiae a protein complex of Mon1 and Ccz1 functions with the small GTPase Ypt7 to mediate vesicle trafficking to the vacuole. Mon1 belongs to DUF254 found in a diverse range of eukaryotic genomes, while Ccz1 includes a CHiPS domain that is also present in a known human protein trafficking disorder gene (HPS-4). The present work identifies the CHiPS domain and a sequence region from another trafficking disorder gene (HPS-1) as homologs of an N-terminal domain from DUF254. This link establishes the evolutionary conservation of a protein complex (HPS-1/HPS-4) that functions similarly to Mon1/Ccz1 in vesicle trafficking to lysosome-related organelles of diverse eukaryotic species. Furthermore, the newly identified DUF254 domain is a distant homolog of the mu-adaptin longin domain found in clathrin adapter protein (AP) complexes of known structure that function to localize cargo protein to specific organelles. In support of this fold assignment, known longin domains such as the AP complex sigma-adaptin, the synaptobrevin N-terminal domains sec22 and Ykt6, and the srx domain of the signal recognition particle receptor also regulate vesicle trafficking pathways by mediating SNARE fusion, recognizing specialized compartments, and interacting with small GTPases that resemble Ypt7.

Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum.

Kamena F, Spang A
Science. 2004; 304: 286-9

Directionality in intracellular trafficking is essential to ensure the correct localization of proteins along the secretory pathway. Here, we found evidence for an active mechanism that prohibited back-fusion of de novo-generated vesicles with their donor compartment. Tip20p is a peripheral membrane protein implicated in consumption of COPI vesicles at the endoplasmic reticulum. However, a specific mutant of TIP20 did not interfere with COPII vesicle generation but allowed these vesicles to fuse back to the endoplasmic reticulum, a process that does not occur normally in the cell.

Oligomeric state and stoichiometry of p24 proteins in the early secretory pathway.

Jenne N, Frey K, Brugger B, Wieland FT
J Biol Chem. 2002; 277: 46504-11

The p24 proteins belong to a highly conserved family of membrane proteins that cycle in the early secretory pathway. They bind to the coat proteins of COPI and COPII vesicles, and are proposed to be involved in vesicle biogenesis, cargo uptake, and quality control, but their precise function is still under debate. Most p24 proteins form hetero-oligomers, essential for their correct localization and stability. Functional insights regarding the mechanisms of their steady state localization and the role of interaction with coat proteins has been hampered by a lack of data on their concentration and state of oligomerization within the endoplasmic reticulum, the intermediate compartment, and Golgi complex. We have determined for all mammalian p24 family members the size of the oligomers formed and their stoichiometric relation in each of these individual organelles. In contrast to earlier reports, we show that individual members exist as dimers and monomers and that the ratio between these two forms depends on both the organelle investigated and the p24 protein. We find unequal quantities, with p23 and p27 building up concentration gradients, ruling out a simple 1:1 stoichiometry. In addition, we show differential cycling of individual p24 members. These data point to a complex and dynamic system of altering dimerizations of the family members.

Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum.

Jackson MR, Nilsson T, Peterson PA
EMBO J. 1990; 9: 3153-62

Several families of transmembrane endoplasmic reticulum (ER) proteins contain retention motifs in their cytoplasmically exposed tails. Mutational analyses demonstrated that two lysines positioned three and four or five residues from the C-terminus represent the retention motif. The introduction of a lysine preceding the lysine that occurs three residues from the terminus of Lyt2 renders this cell surface protein a resident of the ER. Likewise, the appropriate positioning of two lysine residues in a poly-serine sequence confines marker proteins to the ER. Arginines or histidines cannot replace lysines, suggesting that simple charge interactions are not sufficient to explain the retention. The identified consensus motif may serve as a retrieval signal that brings proteins back from a sorting compartment adjacent to the ER.

Ultrastructural characterization of endoplasmic reticulum--Golgi transport containers (EGTC).

Horstmann H, Ng CP, Tang BL, Hong W
J Cell Sci. 2002; 115: 4263-73

Recent observations made in live cells expressing green fluorescent protein (GFP)-tagged cargo markers have demonstrated the existence of large, mobile transport intermediates linking peripheral ER exit sites (ERES) to the perinuclear Golgi. Using a procedure of rapid ethane freezing, we examined ultrastructurally the intermediates involved in ER-Golgi transport of the vesicular stomatitis virus (VSV) G protein. When released at the permissive temperature of 32 degrees C, VSVG is first found to be concentrated in pleiomorphic, membrane-bound structures (of about 0.4 to 1 microm in diameter) with extensive budding profiles. These structures are devoid of COPII components and Golgi markers, but are enriched in COPI, the retrograde cargo ERGIC53, and the tethering protein p115. The structures appear to be able to undergo fusion with the Golgi stack and are tentatively referred to as ER-Golgi transport containers, or EGTCs. VSVG protein exiting the ERES at 15 degrees C is first found in clusters or strings of COPII-containing small vesicles, and morphological analysis indicates that these clusters and strings of COPII vesicles may coalesce by homotypic fusion to form the EGTCs. Together with the large transport containers mediating transport from the trans-Golgi network to the plasma membrane, EGTCs represents an emerging class of large membranous structures mediating anterograde transport between the major stations of the exocytic pathway.

Isoform-selective oligomer formation of Saccharomyces cerevisiae p24 family proteins.

Hirata R, Nihei C, Nakano A
J Biol Chem. 2013; 288: 37057-70

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24alpha, -beta, -gamma, and -delta). Emp24 and Erv25 are the sole members of p24beta and -delta, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Delta). We studied genetic and physical interactions of p24alpha (Erp1, -5, and -6) and gamma (Erp2, -3, and -4). Deletion of the major p24alpha (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Delta phenotype, and the triple mutation gave a full p24Delta phenotype. Similar genetic interactions were observed among the major p24gamma (Erp2) and the other two gamma members. All the p24alpha/gamma isoforms interacted with both p24beta and -delta. Interaction between p24beta and -delta was isoform-selective, and five major alpha/gamma pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant alphabetagammadelta complexes. We also identified Rrt6 as a novel p24delta isoform. Rrt6 shows only limited sequence identity ( approximately 15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional alphabetagammadelta complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.

Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice.

Gong P, Roseman J, Fernandez CG, Vetrivel KS, Bindokas VP, Zitzow LA, Kar S, Parent AT, Thinakaran G
Mol Neurodegener. 2011; 6: 87-87

BACKGROUND: p23 belongs to the highly conserved p24 family of type I transmembrane proteins, which participate in the bidirectional protein transport between the endoplasmic reticulum and Golgi apparatus. Mammalian p23 has been shown to interact with gamma-secretase complex, and modulate secretory trafficking as well as intramembranous processing of amyloid precursor protein in cultured cells. Negative modulation of beta-amyloid production by p23 in cultured cell lines suggested that elevation of p23 expression in neurons might mitigate cerebral amyloid burden. RESULTS: We generated several lines of transgenic mice expressing human p23 in neurons under the control of Thy-1.2 promoter. We found that even a 50% increase in p23 levels in the central nervous system of mice causes post-natal growth retardation, severe neurological problems characterized by tremors, seizure, ataxia, and uncoordinated movements, and premature death. The severity of the phenotype closely correlated with the level of p23 overexpression in multiple transgenic lines. While the number and general morphology of neurons in Hup23 mice appeared to be normal throughout the brain, abnormal non-Golgi p23 localization was observed in a subset of neurons with high transgene expression in brainstem. Moreover, detailed immunofluorescence analysis revealed marked proliferation of astrocytes, activation of microglia, and thinning of myelinated bundles in brainstem of Hup23 mice. CONCLUSIONS: These results demonstrate that proper level of p23 expression is critical for neuronal function, and perturbing p23 function by overexpression initiates a cascade of cellular reactions in brainstem that leads to severe motor deficits and other neurological problems, which culminate in premature death. The neurological phenotype observed in Hup23 mice highlights significant adverse effects associated with manipulating neuronal expression of p23, a previously described negative modulator of gamma-secretase activity and beta-amyloid production. Moreover, our report has broader relevance to molecular mechanisms in several neurodegenerative diseases as it highlights the inherent vulnerability of the early secretory pathway mechanisms that ensure proteostasis in neurons.

p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway.

Gommel D, Orci L, Emig EM, Hannah MJ, Ravazzola M, Nickel W, Helms JB, Wieland FT, Sohn K
FEBS Lett. 1999; 447: 179-85

COPI-coated vesicles that bud off the Golgi complex contain two major transmembrane proteins, p23 and p24. We have localized the protein at the Golgi complex and at COPI-coated vesicles. Transport from the intermediate compartment (IC) to the Golgi can be blocked at 15 degrees C, and under these conditions p24 accumulates in peripheral punctated structures identified as IC. Release from the temperature block leads to a redistribution of p24 to the Golgi, showing that p24, similar to p23, cycles between the IC and Golgi complex. Immunoprecipitations of p24 from cell lysates and from detergent-solubilized Golgi membranes and COPI-coated vesicles show that p24 and p23 interact with each other to form a complex. Transient transfection of p23 in HeLa cells shows that p23 and p24 colocalize in structures induced by the overexpression of p23. Taken together p24 interacts with p23 and constitutively cycles between the organelles of the early secretory pathway.

Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1.

Giraudo CG, Maccioni HJ
Mol Biol Cell. 2003; 14: 3753-66

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

Proteinase-activated receptors (PARs) - focus on receptor-receptor-interactions and their physiological and pathophysiological impact.

Gieseler F, Ungefroren H, Settmacher U, Hollenberg MD, Kaufmann R
Cell Commun Signal. 2013; 11: 86-86

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal "tethered ligand" domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects.In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease.

The Saccharomyces cerevisiae early secretion mutant tip20 is synthetic lethal with mutants in yeast coatomer and the SNARE proteins Sec22p and Ufe1p.

Frigerio G
Yeast. 1998; 14: 633-46

Tip20p is an 80 kDa cytoplasmic protein bound to the cytoplasmic surface of the endoplasmic reticulum (ER) by interaction with the type II integral membrane protein Sec20p. Both proteins are required for vesicular transport between the ER and Golgi complex. Recently, sec20-1 was found to be defective in retrograde transport. A collection of temperature-sensitive tip20 mutants are shown to be lethal in combination with ufe1-1, a target SNARE of the ER and ret2-1, yeast delta-COP. A subset of tip20 mutants was found to be lethal in combination with sec20-1, sec21-1, sec22-3 and sec27-1. Since all pairwise combinations of a tip20 mutant, sec20-1, and ufe1-1 are lethal, Tip20p and Sec20p might be part of the docking complex for Golgi-derived retrograde transport vesicles. Since carboxy-terminal tip20 truncations are lethal in combination with mutants in three coatomer subunits, Tip20p might be involved in binding or uncoating of COPI coated retrograde transport vesicles.

YOS9, the putative yeast homolog of a gene amplified in osteosarcomas, is involved in the endoplasmic reticulum (ER)-Golgi transport of GPI-anchored proteins.

Friedmann E, Salzberg Y, Weinberger A, Shaltiel S, Gerst JE
J Biol Chem. 2002; 277: 35274-81

The OS-9 gene maps to a region (q13-15) of chromosome 12 that is highly amplified in human osteosarcomas and encodes a protein of unknown function. Here we have characterized a homolog designated as YOS9 (YDR057w) from Saccharomyces cerevisiae. The yeast protein (Yos9) is a membrane-associated glycoprotein that localizes to the endoplasmic reticulum (ER). YOS9 interacts genetically with genes involved in ER-Golgi transport, particularly SEC34, whose temperature-sensitive mutant is rescued by YOS9 overexpression. Interestingly, Yos9 appears to play a direct role in the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus. Yos9 binds directly to Gas1 and Mkc7 and accelerates Gas1 transport and processing in cells overexpressing YOS9. Correspondingly, Gas1 processing is slowed in cells bearing a deletion in YOS9. No effect upon the transport and processing of non-GPI-anchored proteins (e.g. invertase and carboxypeptidase Y) was detected in cells either lacking or overexpressing Yos9. As Yos9 is not a component of the Emp24 complex, it may act as a novel escort factor for GPI-anchored proteins in ER-Golgi transport in yeast and possibly in mammals.

Sorting determinants in the transmembrane domain of p24 proteins.

Fiedler K, Rothman JE
J Biol Chem. 1997; 272: 24739-42

Members of the p24 family of putative cargo receptors are proposed to contain retrograde and anterograde trafficking signals in their cytoplasmic domain to facilitate coat protein binding and cycling in the secretory pathway. We have analyzed the role of the transmembrane domain (TMD) of a p24 protein isolated from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi complex involved a glutamic acid residue and a conserved glutamine in the TMD. The TMD glutamic acid mediated the localization of the chimeras to the ER in the absence of the conserved glutamine. Efficient ER exit required the TMD glutamine and was further facilitated by a pair of phenylalanine residues in the cytoplasmic tail. TMD residues of p24 proteins may mediate the interaction with integral membrane proteins of the vesicle budding machinery to ensure p24 packaging into transport vesicles.

Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p.

Fatal N, Karhinen L, Jokitalo E, Makarow M
J Cell Sci. 2004; 117: 1665-73

Exit of proteins from the yeast endoplasmic reticulum (ER) is thought to occur in vesicles coated by four proteins, Sec13p, Sec31p, Sec23p and Sec24p, which assemble at ER exit sites to form the COPII coat. Sec13p may serve a structural function, whereas Sec24p has been suggested to operate in selection of cargo proteins into COPII vesicles. We showed recently that the soluble glycoprotein Hsp150 exited the ER in the absence of Sec13p function. Here we show that its ER exit did not require functional Sec24p. Hsp150 was secreted to the medium in a sec24-1 mutant at restrictive temperature 37 degrees C, while cell wall invertase and vacuolar carboxypeptidase Y remained in the ER. The determinant guiding Hsp150 to this transport route was mapped to the C-terminal domain of 114 amino acids by deletion analysis, and by an HRP fusion protein-based EM technology adapted here for yeast. This domain actively mediated ER exit of Sec24p-dependent invertase in the absence of Sec24p function. However, the domain was entirely dispensable for ER exit when Sec24p was functional. The Sec24p homolog Sfb2p was shown not to compensate for nonfunctional Sec24p in ER exit of Hsp150. Our data show that a soluble cargo protein, Hsp150, is selected actively and specifically to budding sites lacking normal Sec24p by a signature residing in its C-terminal domain.

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

Evan GI, Lewis GK, Ramsay G, Bishop JM
Mol Cell Biol. 1985; 5: 3610-6

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.

Secretion of the type 2 peritrophic matrix protein, peritrophin-15, from the cardia.

Eisemann C, Wijffels G, Tellam RL
Arch Insect Biochem Physiol. 2001; 47: 76-85

The midgut of most insects is lined with a peritrophic matrix, which is thought to facilitate digestion and protect the midgut digestive epithelial cells from abrasive damage and invasion by ingested micro-organisms. The type 2 peritrophic matrix is synthesised by a complex and highly specialised organ called the cardia typically located at the junction of the cuticle-lined foregut and midgut. Although the complex anatomy of this small organ has been described, virtually nothing is known of the molecular processes that lead to the assembly of the type 2 peritrophic matrix in the cardia. As a step towards understanding the synthesis of the peritrophic matrix, the synthesis and secretion of the intrinsic peritrophic matrix protein, peritrophin-15 has been followed in the cardia of Lucilia cuprina larvae using immuno-gold localisations. The protein is synthesised by cardia epithelial cells, which have abundant rough endoplasmic reticulum, Golgi, and vesicles indicative of a general secretory function. Peritrophin-15 is packaged into secretory vesicles probably produced from Golgi and transported to the cytoplasmic face of the apical plasma membrane. The vesicles fuse with the plasma membrane at the base of the microvilli and release peritrophin-15 into the inter-microvilli spaces. The protein then becomes associated with the nascent peritrophic matrix, which lies along the tips of the epithelial cell microvilli. It is proposed that peritrophin-15 binds to the ends of chitin fibrils present in the nascent peritrophic matrix, thereby protecting the fibril from the action of exochitinases.

Archaeal signal peptidases from the genus Thermoplasma: structural and mechanistic hybrids of the bacterial and eukaryal enzymes.

Eichler J
J Mol Evol. 2002; 54: 411-5

Fusogenic domains of golgi membranes are sequestered into specialized regions of the stack that can be released by mechanical fragmentation.

Dominguez M, Fazel A, Dahan S, Lovell J, Hermo L, Claude A, Melancon P, Bergeron JJ
J Cell Biol. 1999; 145: 673-88

A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.

gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer.

Dominguez M, Dejgaard K, Fullekrug J, Dahan S, Fazel A, Paccaud JP, Thomas DY, Bergeron JJ, Nilsson T
J Cell Biol. 1998; 140: 751-65

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

Computer modeling of the membrane interaction of FYVE domains.

Diraviyam K, Stahelin RV, Cho W, Murray D
J Mol Biol. 2003; 328: 721-36

FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways. Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes. Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association. Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated. Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes. Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration. Finally, the computational results are extended to FYVE domains of unknown structure through the construction of high quality homology models for human FYVE sequences.

Use1p is a yeast SNARE protein required for retrograde traffic to the ER.

Dilcher M, Veith B, Chidambaram S, Hartmann E, Schmitt HD, Fischer von Mollard G
EMBO J. 2003; 22: 3664-74

SNAREs on transport vesicles and target membranes are required for vesicle targeting and fusion. Here we describe a novel yeast protein with a typical SNARE motif but with low overall amino acid homologies to other SNAREs. The protein localized to the endoplasmic reticulum (ER) and was therefore named Use1p (unconventional SNARE in the ER). A temperature-sensitive use1 mutant was generated. use1 mutant cells accumulated the ER forms of carboxypeptidase Y and invertase. More specific assays revealed that use1 mutant cells were defective in retrograde traffic to the ER. This was supported by strong genetic interactions between USE1 and the genes encoding SNAREs in retrograde traffic to the ER. Antibodies directed against Use1p co-immunoprecipitated the SNAREs Ufe1p, myc-Sec20p and Sec22p, which form a SNARE complex required for retrograde traffic from the Golgi to the ER, but neither Bos1p nor Bet1p (members of the SNARE complex in anterograde traffic to the Golgi). Therefore, we conclude that Use1p is a novel SNARE protein that functions in retrograde traffic from the Golgi to the ER.

The p24 family member p23 is required for early embryonic development.

Denzel A, Otto F, Girod A, Pepperkok R, Watson R, Rosewell I, Bergeron JJ, Solari RC, Owen MJ
Curr Biol. 2000; 10: 55-8

The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.

Quantitative ER <--> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells.

Dahm T, White J, Grill S, Fullekrug J, Stelzer EH
Mol Biol Cell. 2001; 12: 1481-98

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/- 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF(4)(-) treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.

Computational identification of root hair-specific genes in Arabidopsis.

Cvrckova F, Bezvoda R, Zarsky V
Plant Signal Behav. 2010; 5: 1407-18

Activated cortical domains (ACDs) are regions of the plant cell cortex performing localized membrane turnover, delimited by concerted action of the cortical cytoskeleton and endomembrane compartments. Arabidopsis thaliana rhizodermis consists of two cell types differing by a single ACD (trichoblasts, carrying tip-growing root hairs, and hairless atrichoblasts), providing a model for the study of ACD determination. We compiled a set of genes specifically upregulated in root hairs from published transcriptome data, and compared it with a "virtual Arabidopsis root hair proteome", i.e. a list of computationally identified homologs of proteins from the published soybean root hair proteome. Both data sets were enriched in genes and proteins associated with root hairs in functional studies, but there was little overlap between the transcriptome and the proteome: the former captured gene products specific to root hairs, while the latter selected those abundant in root hairs but not necessarily specific to them. Decisive steps in ACD specification may be performed by signaling proteins of high expression specifity and low abundance. Nevertheless, 73 genes specifically transcribed in Arabidopsis trichoblasts or root hairs encode homologs of abundant root hair proteins from soybean. Most of them encode "housekeeping" proteins required for rapid tip growth. However, among the "candidates" is also a generative actin isoform, ACT11. Preliminary characterization of an act11 mutant allele indeed suggests a hitherto unexpected role for this gene in root and root hair development.

Coatomer interaction with di-lysine endoplasmic reticulum retention motifs.

Cosson P, Letourneur F
Science. 1994; 263: 1629-31

Although signals for retention in the endoplasmic reticulum (ER) have been identified in the cytoplasmic domain of various ER-resident type I transmembrane proteins, the mechanisms responsible for ER retention are still unknown. Yeast and mammalian ER retention motifs interacted specifically in cell lysates with the coatomer, a polypeptide complex implicated in membrane traffic. Mutations that affect the ER retention capacity of the motifs also abolished binding of the coatomer. These results suggest a role for the coatomer in the retrieval of transmembrane proteins to the ER in both yeast and mammals.

A role for Tlg1p in the transport of proteins within the Golgi apparatus of Saccharomyces cerevisiae.

Coe JG, Lim AC, Xu J, Hong W
Mol Biol Cell. 1999; 10: 2407-23

Members of the syntaxin protein family participate in the docking-fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38 degrees C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast alpha-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.

The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum.

Cocquerel L, Duvet S, Meunier JC, Pillez A, Cacan R, Wychowski C, Dubuisson J
J Virol. 1999; 73: 2641-9

Hepatitis C virus (HCV) glycoproteins E1 and E2 assemble to form a noncovalent heterodimer which, in the cell, accumulates in the endoplasmic reticulum (ER). Contrary to what is observed for proteins with a KDEL or a KKXX ER-targeting signal, the ER localization of the HCV glycoprotein complex is due to a static retention in this compartment rather than to its retrieval from the cis-Golgi region. A static retention in the ER is also observed when E2 is expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain (TMD) of E2. Although they do not exclude the presence of an intracellular localization signal in E1, these data do suggest that the TMD of E2 is an ER retention signal for HCV glycoprotein complex. In this study chimeric proteins containing the ectodomain of CD4 or CD8 fused to the C-terminal hydrophobic sequence of E1 were shown to be localized in the ER, indicating that the TMD of E1 is also a signal for ER localization. In addition, these chimeric proteins were not processed by Golgi enzymes, indicating that the TMD of E1 is responsible for true retention in the ER, without recycling through the Golgi apparatus. Together, these data suggest that at least two signals (TMDs of E1 and E2) are involved in ER retention of the HCV glycoprotein complex.

Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

Chomczynski P, Sacchi N
Anal Biochem. 1987; 162: 156-9

A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

Specific interaction between SNAREs and epsin N-terminal homology (ENTH) domains of epsin-related proteins in trans-Golgi network to endosome transport.

Chidambaram S, Mullers N, Wiederhold K, Haucke V, von Mollard GF
J Biol Chem. 2004; 279: 4175-9

SNARE proteins on transport vesicles and target membranes have important roles in vesicle targeting and fusion. Therefore, localization and activity of SNAREs have to be tightly controlled. Regulatory proteins bind to N-terminal domains of some SNAREs. vti1b is a mammalian SNARE that functions in late endosomal fusion. To investigate the role of the N terminus of vti1b we performed a yeast two-hybrid screen. The N terminus of vti1b interacted specifically with the epsin N-terminal homology (ENTH) domain of enthoprotin/CLINT/epsinR. The interaction was confirmed using in vitro binding assays. This complex formation between a SNARE and an ENTH domain was conserved between mammals and yeast. Yeast Vti1p interacted with the ENTH domain of Ent3p. ENTH proteins are involved in the formation of clathrin-coated vesicles. Both epsinR and Ent3p bind adaptor proteins at the trans-Golgi network. Vti1p is required for multiple transport steps in the endosomal system. Genetic interactions between VTI1 and ENT3 were investigated. Synthetic defects suggested that Vti1p and Ent3p cooperate in transport from the trans-Golgi network to the prevacuolar endosome. Our experiments identified the first cytoplasmic protein binding to specific ENTH domains. These results point toward a novel function of the ENTH domain and a connection between proteins that function either in vesicle formation or in vesicle fusion.

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6.

Charest A, Lane K, McMahon K, Housman DE
J Biol Chem. 2001; 276: 29456-65

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.

Six-transmembrane topology for Golgi anti-apoptotic protein (GAAP) and Bax inhibitor 1 (BI-1) provides model for the transmembrane Bax inhibitor-containing motif (TMBIM) family.

Carrara G, Saraiva N, Gubser C, Johnson BF, Smith GL
J Biol Chem. 2012; 287: 15896-905

The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms.

Cardenas J, Rivero S, Goud B, Bornens M, Rios RM
BMC Biol. 2009; 7: 56-56

BACKGROUND: The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. RESULTS: By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. CONCLUSION: We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.

RUN domains: a new family of domains involved in Ras-like GTPase signaling.

Callebaut I, de Gunzburg J, Goud B, Mornon JP
Trends Biochem Sci. 2001; 26: 79-83

RUN domains are present in several proteins that are linked particularly to the functions of GTPases in the Rap and Rab families. They could hence play an important role in multiple Ras-like GTPase signaling pathways.

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles.

Baudhuin P, Evrard P, Berthet J
J Cell Biol. 1967; 32: 181-91

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 micro) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.

The organization of endoplasmic reticulum export complexes.

Bannykh SI, Rowe T, Balch WE
J Cell Biol. 1996; 135: 19-35

Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI.

Getting into the Golgi.

Bannykh SI, Nishimura N, Balch WE
Trends Cell Biol. 1998; 8: 21-5

The intermediate compartment residing between the endoplasmic reticulum (ER) and the Golgi is now recognized to be a dynamic structure that captures cargo released from the ER in COPII vesicular carriers and promotes recycling by COPI vesicular carriers. These and other findings now provide compelling evidence for the importance of this intermediate in balancing anterograde and retrograde flow through the early secretory pathway and in the formation and maintenance of the Golgi stack.

Early stages of Golgi vesicle and tubule formation require diacylglycerol.

Asp L, Kartberg F, Fernandez-Rodriguez J, Smedh M, Elsner M, Laporte F, Barcena M, Jansen KA, Valentijn JA, Koster AJ, Bergeron JJ, Nilsson T
Mol Biol Cell. 2009; 20: 780-90

We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed.

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ
Nucleic Acids Res. 1997; 25: 3389-402

The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

[Current data on the secretory function of the Golgi complex].

Alov IA
Usp Sovrem Biol. 1968; 65: 144-51

A microtubule-binding protein associated with membranes of the Golgi apparatus.

Allan VJ, Kreis TE
J Cell Biol. 1986; 103: 2229-39

A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins.