Secondary literature sources for the RAB domain

For each of the hand selected primary papers associated with the RAB 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.

Rab GTPases in vesicular transport.

Zerial M, Stenmark H
Curr Opin Cell Biol. 1993; 5: 613-20

Specificity and directionality are two features shared by the numerous steps of membrane transport that connect cellular organelles. By shuttling between specific membrane compartments and the cytoplasm, small GTPases of the Rab family appear to regulate membrane traffic in a cyclical manner. The restriction of certain Rab proteins to differentiated cell types supports a role for these GTPases in defining the specificity of membrane trafficking.

Rab proteins as membrane organizers.

Zerial M, McBride H
Nat Rev Mol Cell Biol. 2001; 2: 107-17

Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport. Rab proteins and their effectors coordinate consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles to their target compartment. These molecules are highly compartmentalized in organelle membranes, making them excellent candidates for determining transport specificity and organelle identity.

[Vesicle-mediated protein transport system]

Yoshimura T
Tanpakushitsu Kakusan Koso. 1993; 38: 1152-9

New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion.

Wurmser AE, Sato TK, Emr SD
J Cell Biol. 2000; 151: 551-62

The class C subset of vacuolar protein sorting (Vps) proteins (Vps11, Vps18, Vps16 and Vps33) assembles into a vacuole/prevacuole-associated complex. Here we demonstrate that the class C-Vps complex contains two additional proteins, Vps39 and Vps41. The COOH-terminal 148 amino acids of Vps39 direct its association with the class C-Vps complex by binding to Vps11. A previous study has shown that a large protein complex containing Vps39 and Vps41 functions as a downstream effector of the active, GTP-bound form of Ypt7, a rab GTPase required for the fusion of vesicular intermediates with the vacuole (Price, A., D. Seals, W. Wickner, and C. Ungermann. 2000. J. Cell Biol. 148:1231-1238). Here we present data that indicate that this complex also functions to stimulate nucleotide exchange on Ypt7. We show that Vps39 directly binds the GDP-bound and nucleotide-free forms of Ypt7 and that purified Vps39 stimulates nucleotide exchange on Ypt7. We propose that the class C-Vps complex both promotes Vps39-dependent nucleotide exchange on Ypt7 and, based on the work of Price et al., acts as a Ypt7 effector that tethers transport vesicles to the vacuole. Thus, the class C-Vps complex directs multiple reactions during the docking and fusion of vesicles with the vacuole, each of which contributes to the overall specificity and efficiency of this transport process.

Formation of secretory vesicles in the biosynthetic pathway.

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

A Rab GTPase is required for homotypic assembly of the endoplasmic reticulum.

Turner MD, Plutner H, Balch WE
J Biol Chem. 1997; 272: 13479-83

To define the requirements for the homotypic fusion of mammalian endoplasmic reticulum (ER) membranes, we have developed a quantitative in vitro enzyme-linked immunosorbent assay. This assay measures the formation of IgG (H2L2) following the fusion of ER microsomes containing either the heavy or light chain subunits. Guanine nucleotide dissociation inhibitor (GDI), a protein that extracts Rab GTPases in the GDP-bound form from membranes, potently inhibits fusion. Inhibition was not observed using GDI mutants defective in Rab binding. Kinetic analysis of the inhibitory effects of GDI revealed that Rab activation is required immediately preceding or coincident with fusion and that this step is preceded by a priming event requiring a member of the AAA ATPase family. Our results suggest that homotypic fusion of ER membranes requires Rab and that Rab activation is a transient event necessary for the formation of a fusion pore leading to the mixing of luminal contents of ER microsomes.

A comparison of yeast ribosomal protein gene DNA sequences.

Teem JL, Abovich N, Kaufer NF, Schwindinger WF, Warner JR, Levy A, Woolford J, Leer RJ, van Raamsdonk-Duin MM, Mager WH, et al.
Nucleic Acids Res. 1984; 12: 8295-312

The DNA sequences of eight yeast ribosomal protein genes have been compared for the purpose of identifying homologous regions which may be involved in the coordinate regulation of ribosomal protein synthesis. A 12 bp homology was identified in the 5' DNA sequence preceding the structural gene for 6 out of 8 yeast ribosomal protein genes. In each case the homologous sequence was found at a position approximately 300 bp preceding the transcription start of the ribosomal protein gene. This homology was not identified in any non-ribosomal protein gene examined. Additional homologies between ribosomal protein genes were identified in the transcribed regions, including the untranslated 5' and 3' DNA regions flanking the coding regions.

APL1, a yeast gene encoding a putative permease for basic amino acids.

Sychrova H, Chevallier MR
Yeast. 1994; 10: 653-7

A Saccharomyces cerevisiae gene (1722 bp), encoding a protein (574 aa) highly homologous to the basic-amino-acid permeases LYP1 and CAN1, was sequenced. The gene, which was named APL1 (Amino-acid Permase Like), is located 881 bp upstream from LYP1 (lysine-specific permease), and in head-to-head orientation to it. These sequence data have been deposited in the EMBL/GenBank/DDBJ nucleotide sequence data libraries under Accession Number X74069.

Regulation of vesicular transport by GTP-binding proteins.

Stow JL
Curr Opin Nephrol Hypertens. 1995; 4: 421-5

Intracellular protein trafficking occurs in a series of transport vesicles. Vesicle trafficking is regulated both by heterotrimeric and monomeric GTP-binding proteins (G proteins). Recent studies have explored effector systems used by heterotrimeric G proteins and by monomeric ADP-ribosylation factor G proteins for regulation of vesicle budding. New members of the Rab monomeric G protein family have been identified in polarized cells and new evidence confirms the function of Rab proteins in vesicle targeting. From these data we can begin to reconstruct the signal transduction pathways that regulate intracellular transport.

The Rab GTPase family.

Stenmark H, Olkkonen VM
Genome Biol. 2001; 2: 3007-3007

SUMMARY: The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.

Phosphorylation of GDI and membrane cycling of rab proteins.

Steele-Mortimer O, Gruenberg J, Clague MJ
FEBS Lett. 1993; 329: 313-8

Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co-immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two-dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.

Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange.

Soldati T, Shapiro AD, Svejstrup AB, Pfeffer SR
Nature. 1994; 369: 76-8

The Rab GTPases are key regulators of vesicular transport. A fraction of Rab proteins is present in the cytosol, bound with GDP, complexed to a protein termed GDI. Rab9 is localized primarily to late endosomes, where it aids the transport of mannose 6-phosphate receptors to the trans-Golgi network. It has been proposed that Rab proteins are delivered to specific membranes by GDI, and that this process is accompanied by the exchange of bound GDP for GTP. In addition, Rab localization requires carboxy-terminal prenylation and specific structural determinants. Here we describe the reconstitution of the selective targeting of prenylated Rab9 protein onto late endosome membranes and show that this process is accompanied by endosome-triggered nucleotide exchange.

Rab proteins and the road maps for intracellular transport.

Simons K, Zerial M
Neuron. 1993; 11: 789-99

Mechanism of digeranylgeranylation of Rab proteins. Formation of a complex between monogeranylgeranyl-Rab and Rab escort protein.

Shen F, Seabra MC
J Biol Chem. 1996; 271: 3692-8

Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates.

Ypt/rab gtpases: regulators of protein trafficking.

Segev N
Sci STKE. 2001; 2001: 11-11

Ypt/Rab guanosine triphosphatases (GTPases) have emerged in the last decade as key regulators of protein transport in all eukaryotic cells. They seem to be involved in all aspects of vesicle trafficking: vesicle formation, motility, and docking, and membrane remodeling and fusion. The functions of Ypt/Rabs are themselves controlled by upstream regulators that stimulate both their nucleotide cycling and their cycling between membranes. Ypt/Rabs transmit signals to downstream effectors in a guanosine triphosphate (GTP)-dependent manner. The identity of upstream regulators and downstream effectors is known for a number of Ypt/Rabs, and models for their mechanisms of action are emerging. In at least two cases, Ypt/Rab upstream regulators and downstream effectors are found together in a single complex. In agreement with the idea that Ypt/Rabs function in all aspects of vesicular transport, their diverse effectors have recently been shown to function in all identified aspects of vesicle transport. Activators and effectors for individual Ypt/Rabs share no similarity, but are conserved between yeast and mammalian cells. Finally, cross talk demonstrated among the various Ypt/Rabs, and between Ypt/Rabs and other signaling factors, suggests possible coordination among secretory steps, as well as between protein transport and other cellular processes.

Ypt and Rab GTPases: insight into functions through novel interactions.

Segev N
Curr Opin Cell Biol. 2001; 13: 500-11

Ypt/Rab GTPases are key regulators of vesicular transport in eukaryotic cells. During the past two years, a number of new Ypt/Rab-interacting proteins have been identified and shown to serve as either upstream regulators or downstream effectors. Proteins that interact with these regulators and effectors of Ypt/Rabs have also been identified, and together they provide new insights into Ypt/Rab mechanisms of action. The picture that emerges from these studies suggests that Ypt/Rabs function in multiple and diverse aspects of vesicular transport. In addition, not only are Ypt/Rabs highly conserved, but their functions and interactions are as well. Interestingly, crosstalk among Ypt/Rabs and between Ypt/Rabs and other signaling factors, suggest the possibility of coordination of the individual vesicular transport steps and of the protein transport machinery with other cellular processes.

Coated vesicles: a diversity of form and function.

Schmid SL, Damke H
FASEB J. 1995; 9: 1445-53

In every well-characterized example, the small transport vesicles that mediate membrane trafficking between intracellular organelles are encased in a protein coat. In general, the coat proteins assemble from cytosolic pools onto the membrane and play a critical role in vesicle formation. Recent reviews have emphasized the clear similarities in the mechanisms that drive vesicle budding at distinct cellular locations. Here we focus on the diversity of solutions to an apparently related biological task. These mechanistic differences are likely to be physiologically important determinants of the diversity in form, and function of coated transport vesicles.

Rab GTPases, directors of vesicle docking.

Schimmoller F, Simon I, Pfeffer SR
J Biol Chem. 1998; 273: 22161-4

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Schatz PJ, Pillus L, Grisafi P, Solomon F, Botstein D
Mol Cell Biol. 1986; 6: 3711-21

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

Implications of the SNARE hypothesis for intracellular membrane topology and dynamics.

Rothman JE, Warren G
Curr Biol. 1994; 4: 220-33

The SNARE hypothesis provides a mechanism for the specific docking and fusion of transport vesicles with their target membranes. A simple extension of the hypothesis can explain many cellular processes, including the stacking of Golgi cisternae, retrograde transport and homotypic fusion; it can also explain the morphology of intracellular membranes and their dynamics during mitosis.

Throttles and dampers: controlling the engine of membrane fusion.

Rothman JE, Sollner TH
Science. 1997; 276: 1212-3

The protein machinery of vesicle budding and fusion.

Rothman JE
Protein Sci. 1996; 5: 185-94

A general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins.

Powers S, Kataoka T, Fasano O, Goldfarb M, Strathern J, Broach J, Wigler M
Cell. 1984; 36: 607-12

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.

Selective membrane recruitment of Rab GTPases.

Pfeffer SR, Soldati T, Geissler H, Rancano C, Dirac-Svejstrup B
Cold Spring Harb Symp Quant Biol. 1995; 60: 221-7

Rab GTPases: master regulators of membrane trafficking.

Pfeffer SR
Curr Opin Cell Biol. 1994; 6: 522-6

Rab GTPases are thought to be likely to catalyze the accurate association of pairs of targeting molecules located on the surfaces of transport vesicles with their corresponding membrane acceptors. Advances during the past year have solidified our understanding of the mechanisms by which Rab proteins are recruited onto nascent transport vesicles and retrieved from their fusion targets. Functional analyses of Rab proteins in living cells have led to the surprising observation that vesicles do not seem to form if the appropriate Rab protein, in its GTP-bound conformation, is not present.

Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease.

Pereira-Leal JB, Hume AN, Seabra MC
FEBS Lett. 2001; 498: 197-200

Small GTPases of the Rab family regulate membrane transport pathways. More than 50 mammalian Rab proteins are known, many with transport step-specific localisation. Rabs must associate with cellular membranes for activity and membrane attachment is mediated by prenyl (geranylgeranyl) post-translational modification. Mutations in genes encoding proteins essential for the geranylgeranylation reaction, Rab escort protein and Rab geranylgeranyl transferase, underlie genetic diseases. Choroideremia patients have loss of function mutations in REP1 and the murine Hermansky-Pudlak syndrome model gunmetal possesses a splice-site mutation in the alpha-subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases.

Vesicular transport with emphasis on exocytosis.

Park CS, Lee PH
Yonsei Med J. 1994; 35: 355-77

The eukaryotic cell is compartmentalized by a series of vesicular organelles which constitute the endocytic and exocytic transport pathways. Each vesicular compartment has distinct sets of membrane proteins, structures and functions. Despite continuous vesicular transport, each vesicular compartment maintains its structure and function by use of retention and retrieval signal for its own resident proteins. Proteins in transit along the endocytic and exocytic pathway are transported without admixing with cytoplasmic constituents by successive steps of budding from the donor vesicles, formation of intermediate transport vesicles, transport, targeting to and fusion with acceptor vesicles. Specificity and fidelity of the vesicular transport are conferred by vesicular membrane proteins and small molecular weight GTP-binding proteins of the Rab subfamily. Proteins for export are packaged into specific vesicles for their final destinations. Insertion into and retrieval from the plasma membrane of transport proteins in response to cellular stimulus are a new paradigm of cellular regulatory mechanism. Secretion of neurotransmitters, hormones and enzymes by exocytosis involves a complex set of cytosolic proteins, G-proteins, proteins on the secretory granule membrane and plasma membrane. Much progress has been recently made in identifying proteins and factors involved in the exocytosis. But the molecular interactions among identified proteins and regulatory factors are unknown and remain to be elucidated. Finally our chemiosmotic hypothesis which involves the H+ electrochemical gradient across the secretory granule membrane generated by an ATP-dependent electrogenic H(+)-ATPase as the potential driving force for fusion and release of granule contents will be discussed.

Role of Rab GTPases in membrane traffic.

Olkkonen VM, Stenmark H
Int Rev Cytol. 1997; 176: 1-85

Small GTPases of the Rab subfamily have been known to be key regulators of intracellular membrane traffic since the late 1980s. Today this protein group amounts to more than 40 members in mammalian cells which localize to distinct membrane compartments and exert functions in different trafficking steps on the biosynthetic and endocytic pathways. Recent studies indicate that cycles of GTP binding and hydrolysis by the Rab proteins are linked to the recruitment of specific effector molecules on cellular membranes, which in turn impact on membrane docking/fusion processes. Different Rabs may, nevertheless, have slightly different principles of action. Studies performed in yeast suggest that connections between the Rabs and the SNARE machinery play a central role in membrane docking/fusion. Further elucidation of this linkage is required in order to fully understand the functional mechanisms of Rab GTPases in membrane traffic.

GTPases: multifunctional molecular switches regulating vesicular traffic.

Nuoffer C, Balch WE
Annu Rev Biochem. 1994; 63: 949-90

Vesicular transport. No exchange without receipt.

Novick P, Garrett MD
Nature. 1994; 369: 18-9

Friends and family: the role of the Rab GTPases in vesicular traffic.

Novick P, Brennwald P
Cell. 1993; 75: 597-601

Isolation and sequence of the gene for actin in Saccharomyces cerevisiae.

Ng R, Abelson J
Proc Natl Acad Sci U S A. 1980; 77: 3912-6

The yeast Saccharomyces cerevisiae is known to contain the highly conserved and unbiquitous protein actin. We have used cloned actin sequences from Dictyostelium discoideum to identify and clone the actin gene in yeast. Hybridization to genomic fragments of yeast DNA suggest that there is a single actin gene in yeast. We have determined the nucleotide sequence of that gene and its flanking regions. The sequence of the gene reveals an intervening sequence of 309 base pairs in the coding sequences at the 5' end of the gene. The existence and location of the intervening sequence was verified by using the dideoxy chain termination technique to determine the sequence at the 5' terminus of the actin mRNA. The similarity of the splice junction sequences in this gene to those found in higher eukaryotes suggests that yeast must possess a similar splicing enzyme.

A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

Miyamoto S, Ohya Y, Sano Y, Sakaguchi S, Iida H, Anraku Y
Biochem Biophys Res Commun. 1991; 181: 604-10

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.

A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits.

McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E
Curr Biol. 1998; 8: 34-45

BACKGROUND: Clathrin-coated pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. RESULTS: We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-coated pits. CONCLUSIONS: We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.

Rab proteins.

Martinez O, Goud B
Biochim Biophys Acta. 1998; 1404: 101-12

Rab proteins form the largest branch of the Ras superfamily of GTPases. They are localized to the cytoplasmic face of organelles and vesicles involved in the biosynthetic/secretory and endocytic pathways in eukaryotic cells. It is now well established that Rab proteins play an essential role in the processes that underlie the targeting and fusion of transport vesicles with their appropriate acceptor membranes. However, the recent discovery of several putative Rab effectors, which are not related to each other and which fulfil diverse functions, suggests a more complex role for Rab proteins. At least two Rab proteins act at the level of the Golgi apparatus. Rab1 and its yeast counterpart Ypt1 control transport events through early Golgi compartments. Work from our laboratory points out a role for Rab6 in intra-Golgi transport, likely in a retrograde direction.

Prenylation-dependent interaction of Rab proteins with GDP dissociation inhibitors.

Maltese WA, Wilson AL, Erdman RA
Biochem Soc Trans. 1996; 24: 703-8

The gene LEO1 on yeast chromosome XV encodes a non-essential, extremely hydrophilic protein.

Magdolen V, Lang P, Mages G, Hermann H, Bandlow W
Biochim Biophys Acta. 1994; 1218: 205-9

A 5.6 kbp segment of DNA from Saccharomyces cerevisiae chromosome XV has been isolated and sequenced. Genetic and nucleotide sequence analyses revealed that this region is closely linked to the ADE2 marker on chromosome XV and densely packed with genetic information. We show the gene organization of the entire region and report the nucleotide sequence of the gene, LEO1, which occurs in single copy in the haploid genome. The deduced amino acid sequence specifies an extremely hydrophilic protein with pronounced domain structure (molecular mass 53.9 kDa). The gene is constitutively expressed at a low level and is non-essential, as indicated by the absence of a phenotype from gene disruption mutants.

Bacterial cell division and the Z ring.

Lutkenhaus J, Addinall SG
Annu Rev Biochem. 1997; 66: 93-116

Bacterial cell division occurs through the formation of an FtsZ ring (Z ring) at the site of division. The ring is composed of the tubulin-like FtsZ protein that has GTPase activity and the ability to polymerize in vitro. The Z ring is thought to function in vivo as a cytoskeletal element that is analogous to the contractile ring in many eukaryotic cells. Evidence suggests that the Z ring is utilized by all prokaryotic organisms for division and may also be used by some eukaryotic organelles. This review summarizes our present knowledge about the formation, function, and evolution of the Z ring in prokaryotic cell division.

t-SNARE activation through transient interaction with a rab-like guanosine triphosphatase.

Lupashin VV, Waters MG
Science. 1997; 276: 1255-8

Intracellular vesicle targeting involves the interaction of vesicle proteins, termed v-SNAREs, with target membrane proteins, termed t-SNAREs. Assembly of v-SNARE-t-SNARE targeting complexes is modulated by members of the Sec1-Sly1 protein family, and by small guanosine triphosphatases termed Rabs. The interactions of these proteins during assembly of the endoplasmic reticulum-to-Golgi targeting complex in Saccharomyces cerevisiae were studied. The data suggest that the Rab protein Ypt1p transiently interacts with the t-SNARE Sed5p and results in displacement of the negative regulator Sly1p, allowing subsequent formation of the v-SNARE-t-SNARE targeting complex.

Molecular dissection of guanine nucleotide dissociation inhibitor function in vivo. Rab-independent binding to membranes and role of Rab recycling factors.

Luan P, Balch WE, Emr SD, Burd CG
J Biol Chem. 1999; 274: 14806-17

Guanine nucleotide dissociation inhibitor (GDI) is an essential protein required for the recycling of Rab GTPases mediating the targeting and fusion of vesicles in the exocytic and endocytic pathways. Using site-directed mutagenesis of yeast GDI1, we demonstrate that amino acid residues required for Rab recognition in vitro are critical for function in vivo in Saccharomyces cerevisiae. Analysis of the effects of Rab-binding mutants on function in vivo reveals that only a small pool of recycling Rab protein is essential for growth, and that the rates of recycling of distinct Rabs are differentially sensitive to GDI. Furthermore, we find that membrane association of Gdi1p is Rab-independent. Mutant Gdi1 proteins unable to bind Rabs were able to associate with cellular membranes as efficiently as wild-type Gdi1p, yet caused a striking loss of the endogenous cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when expressed at levels of the normal, endogenous pool. These results demonstrate a potential role for a new recycling factor in the retrieval of Rab-GDP from membranes, and illustrate the importance of multiple effectors in regulating GDI function in Rab delivery and retrieval from membranes.

Exocytosis in excitable cells: a conserved molecular machinery from yeast to neuron.

Lledo PM
Eur J Endocrinol. 1997; 137: 1-9

One of the basic cellular functions of nearly every cell type is the exocytotic release of synthesized molecules, stored and packaged into intracellular vesicles or granules. A variety of approaches has been used to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway. The findings obtained with these approaches suggest that common mechanisms may underlie a wide variety of vesicle-mediated transport steps. This review presents some of the recent findings regarding the study of the cellular mechanisms which control neurotransmitter and hormone release from neurons and endocrine cells respectively, and focuses on regulation of these mechanisms. The similarities between these two cell types can be seen as evidence to support the hypothesis according to which the regulated exocytosis apparatus could have evolved from a constitutive fusion machinery to which some key modulators have been added. Insight into secretory vesicles will be relevant not only to the understanding of vesicular trafficking or cell polarity but also to the understanding of higher nervous functions resulting from synaptic plasticity.

Ypt1p implicated in v-SNARE activation.

Lian JP, Stone S, Jiang Y, Lyons P, Ferro-Novick S
Nature. 1994; 372: 698-701

Synaptobrevin-like membrane proteins that reside on transport vesicles, called the vesicle SNARE (v-SNARE), play a key role in ensuring that a vesicle targets and fuses with its correct acceptor compartment. Here we show that Bos1p, the v-SNARE of yeast endoplasmic reticulum-to-Golgi transport vesicles, pairs with another integral membrane protein of similar topology (Sec22p) on vesicles. This pairing, which appears to require functional Ypt1p (Rab in mammalian cells), may aid the activity of Bos1p on this compartment. These findings suggest that Rabs regulate the specificity of membrane fusion by selectively activating the v-SNARE on carrier vesicles. Because the v-SNARE resides on more than one membrane, such a regulated activation step may be necessary to prevent the premature fusion of donor and acceptor compartments.

Characterization of an ADP-ribosylation factor-like 1 protein in Saccharomyces cerevisiae.

Lee FJ, Huang CF, Yu WL, Buu LM, Lin CY, Huang MC, Moss J, Vaughan M
J Biol Chem. 1997; 272: 30998-1005

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Several ARF-like proteins (ARLs) have been cloned from Drosophila, rat, and human; however, the biological functions of ARLs are unknown. We have identified a yeast gene (ARL1) encoding a protein that is structurally related (>60% identical) to human, rat, and Drosophila ARL1. Biochemical analyses of purified recombinant yeast ARL1 (yARL1) protein revealed properties similar to those ARF and ARL1 proteins, including the ability to bind and hydrolyze GTP. Like other ARLs, recombinant yARL1 protein did not stimulate cholera toxin-catalyzed auto-ADP-ribosylation. yARL1 was not recognized by antibodies against mammalian ARLs or yeast ARFs. Anti-yARL1 antibodies did not cross-react with yeast ARFs, but did react with human ARLs. On subcellular fractionation, yARL1, similar to yARF1, was localized to the soluble fraction. The amino terminus of yARL1, like that of ARF, was myristoylated. Unlike Drosophila Arl1, yeast ARL1 was not essential for cell viability. Like rat ARL1, yARL1 might be associated in part with the Golgi complex. However, yARL1 was not required for endoplasmic reticulum-to-Golgi protein transport, and it may offer an opportunity to define an ARL function in another kind of vesicular trafficking, such as the regulated secretory pathway.

[COP-coated vesicles in intracellular protein transport]

Kuge O, Kuge S
Tanpakushitsu Kakusan Koso. 1995; 40: 2427-35

Identification and characterization of a yeast gene encoding an adenylate kinase homolog.

Konrad M
Biochim Biophys Acta. 1993; 1172: 12-6

Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted in the isolation of a homolog of the previously characterized ADK1. The derived protein sequence is most closely related to mammalian GTP:AMP phosphotransferase (adenylate kinase isozyme 3; AK3); this novel gene is therefore named ADK3. Its deletion from the yeast genome does not lead to an observable change in cellular phenotype. A strain defective for both ADK1 and ADK3 is viable. When introduced on a multicopy plasmid into an ADK1-deficient yeast strain, which shows a reduced proliferation rate, ADK3 did not rescue this growth defect. The protein was also highly overexpressed in E. coli cells. However, no change in enzymatic activity was detected in cellular extracts of yeast or bacteria.

Nucleotide sequence of the GDS1 gene of Saccharomyces cerevisiae.

Konopinska A, Szczesniak B, Boguta M
Yeast. 1995; 11: 1513-8

We have cloned and sequenced the GDS1 gene located on the right arm of chromosome XV of Saccharomyces cerevisiae. The gene codes for a 522 amino acid serine-rich protein with no obvious homology to proteins in the database. GDS1 gene was isolated as the multicopy suppressor of the glycerol-deficient phenotype caused by the nam9-1 mutation in the yeast nuclear gene encoding the mitochondrial ribosomal protein homologous to S4 proteins from various organisms. Disruption-deletion of the GDS1 open reading frame leads to a partial impairment of growth on medium containing glycerol as the carbon source, indicating mitochondrial function of the gene product.

Vesicle formation: dynamic dynamin lives up to its name.

Kirchhausen T
Curr Biol. 1998; 8: 7924-7924

The GTP-binding protein dynamin was initially thought to be required for just the final stages of clathrin-dependent vesicle formation, but recent results indicate that it can actually catalyse many of the essential steps in the vesiculation pathway.

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif.

Joberty G, Tavitian A, Zahraoui A
FEBS Lett. 1993; 330: 323-8

Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.

Rab1a and multiple other Rab proteins are associated with the transcytotic pathway in rat liver.

Jin M, Saucan L, Farquhar MG, Palade GE
J Biol Chem. 1996; 271: 30105-13

To better understand the function of Rab1a, we have immunoisolated Rab1a-associated transport vesicles from rat liver using affinity-purified anti-Rab1a-coated magnetic beads. A fraction enriched in endoplasmic reticulum (ER) to Golgi transport vesicles (CV2, rho = 1.158) was subjected to immunoisolation, and proteins of the bound and non-bound subfractions were analyzed by Western blotting. To our surprise, we found that immunoisolated vesicles contained not only ER markers (105-kDa form of the polymeric IgA receptor (pIgAR)) but also transcytotic markers (dIgA and the 120-kDa form of pIgAR), suggesting that Rab1a is associated with transcytotic vesicles in rat liver. To investigate this possibility, we used an antibody to the cytoplasmic domain of pIgAR to immunoisolate transcytotic vesicles from a fraction (CV1, rho = 1. 146) known to be enriched in these vesicles. Rab1a was detected in the immunoadsorbed subfractions. The composition of the vesicles immunoisolated from the CV1 fraction on anti-Rab1a was similar to that of transcytotic vesicles immunoisolated from the same fraction on pIgAR. Both were enriched in transcytotic markers and depleted in ER and Golgi markers. The main difference between the two was that those isolated on anti-Rab1a appeared to be enriched in postendosomal transcytotic vesicles, whereas those isolated on pIgAR contained both pre- and postendosomal elements. Analysis of anti-Rab1a isolated vesicles using [alpha-32P]GTP overlay demonstrated the presence of multiple GTP-binding proteins. Some of these were identified by immunoblotting as epithelia-specific Rab17 and ubiquitous Rabs1b, -2, and -6. Taken together, these results indicate that: 1) Rab1a is associated with both ER to Golgi and postendosomal transcytotic vesicles, and 2) multiple GTP-binding proteins are associated with each class of isolated vesicle.

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

Huber LA, Dupree P, Dotti CG
Mol Cell Biol. 1995; 15: 918-24

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

Small G proteins of two green algae are localized to exocytic compartments and to flagella.

Huber H, Beyser K, Fabry S
Plant Mol Biol. 1996; 31: 279-93

The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo- and exocytosis in mammalian cells and yeast. Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization. The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes. Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region. Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy. In contrast, we found Ypt1p associated with the contractile vacuole in both V. carteri and C. reinhardtii. Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells. This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae. While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.

The ras-related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast.

Haubruck H, Prange R, Vorgias C, Gallwitz D
EMBO J. 1989; 8: 1427-32

The protein-coding region of the essential Saccharomyces cerevisiae YPT1 gene coding for a ras-related, guanine-nucleotide-binding protein was exchanged in chromosome VI by the protein-coding segment of either the mouse ypt1 gene or the v-Ki-ras gene, and different chimeric YPT1-v-Ki-ras genes. The mouse ypt1 protein with 71% of identical residues compared with the yeast Ypt1 protein could functionally fully replace its yeast homologue as long as the mouse gene was overexpressed under transcriptional control of the inducible GAL10 promoter. In contrast, neither the viral Ki-ras nor the hybrid proteins were able to substitute for the loss of YPT1 gene function. This study suggests that different parts of the yeast Ypt1 protein are required for the interaction with cellular targets and that these essential parts are conserved in the mammalian ypt1 protein.

An essential gene of Saccharomyces cerevisiae coding for an actin-related protein.

Harata M, Karwan A, Wintersberger U
Proc Natl Acad Sci U S A. 1994; 91: 8258-62

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT1 (coding for the filament actin), and a so-called actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (M(r) = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophilic [corrected] segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3. Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in budding yeast that obviously provide different essential functions for survival.

Regulation of intracellular membrane transport.

Gruenberg J, Clague MJ
Curr Opin Cell Biol. 1992; 4: 593-9

A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.

YMC1, a yeast gene encoding a new putative mitochondrial carrier protein.

Graf R, Baum B, Braus GH
Yeast. 1993; 9: 301-5

We have cloned and sequenced a Saccharomyces cerevisiae gene coding for a protein with significant similarities to the mitochondrial carrier family. The gene we termed YMC1 (yeast mitochondrial carrier) is located on chromosome XVI, closely downstream of ARO7 encoding chorismate mutase.

Cloning and characterization of a dominant-negative vps1 allele of the yeast Saccharomyces cerevisiae.

Finken-Eigen M, Muller S, Kohrer K
Biol Chem. 1997; 378: 1187-91

The gene product of the yeast VPS1 gene is a member of a family of high-molecular-weight GTP-binding proteins that are involved in diverse cellular processes. The Vps1 protein (Vps1p) was shown to perform an essential function in the yeast secretory pathway. Here, we report the isolation and characterization of a mutant allele of the VPS1 gene, causing a dominant-negative vacuolar protein sorting (vps) defect, as demonstrated by the mislocalization of the vacuolar hydrolase carboxypeptidase Y (CPY). DNA sequence analysis of the mutant vps1 allele (vps1d-293) revealed a single point mutation, resulting in an amino acid exchange at position 293 from Ala to Asp. The mutation is located downstream of the tripartite GTP-binding motif found in the amino-terminal half of the protein. The observation that expression of wild-type Vps1p partially suppressed the dominant-negative CPY sorting phenotype indicates competition of a non-functional mutant Vps1 protein and a functional wild-type VPS1p for a Vps1p-binding site of an as yet unknown vacuolar protein sorting factor.

The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles.

Elkind NB, Walch-Solimena C, Novick PJ
J Cell Biol. 2000; 149: 95-110

Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.

Removal of Rab GTP-binding proteins from Golgi membranes by GDP dissociation inhibitor inhibits inter-cisternal transport in the Golgi stacks.

Elazar Z, Mayer T, Rothman JE
J Biol Chem. 1994; 269: 794-7

Rab proteins are a family of Ras-like GTPases involved in intracellular membrane traffic. Rab GDI, a cytosolic protein which inhibits the dissociation of GDP from various Rab proteins, is required to maintain a pool of Rab proteins in the cytosol. We describe the purification of a cytosolic factor from bovine liver that inhibits intra-cisternal transport between the Golgi stacks. We identify this factor as a Rab GDI. Half-maximal inhibition of transport was observed in the presence of the same concentration of GDI that is required for removal of Rab proteins from the Golgi.

Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport.

Dumanchin C, Czech C, Campion D, Cuif MH, Poyot T, Martin C, Charbonnier F, Goud B, Pradier L, Frebourg T
Hum Mol Genet. 1999; 8: 1263-9

Presenilin 1 (PS1) mutations account for the majority of early-onset dominant cases of familial Alzheimer's disease. Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticulum, Golgi apparatus, nuclear region and vesicular structures. These proteins include from seven to nine putative transmembrane domains, with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm. We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11, a small GTPase belonging to the Ras-related superfamily. Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11, a region including the fourth GTP-binding domain. Considering the implication of Rab proteins in vesicular transport pathways, the PS-Rab11 inter-action suggests that PSs might be involved in amyloid precursor protein vesicular routing.

Sequence of a novel plant ras-related cDNA from Pisum sativum.

Drew JE, Bown D, Gatehouse JA
Plant Mol Biol. 1993; 21: 1195-9

A clone isolated from a purple podded pea (Pisum sativum L.) cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the ras superfamily. The ras superfamily are a group of monomeric GTP-binding proteins of 21-25 kDa found in eukaryotic cells. Conserved sequences in the isolated clone include the GTP-binding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. Comparisons of the predicted amino acid sequence with those of other ras proteins show significantly higher homologies (ca. 70%) to two mammalian gene products, those of the BRL-ras oncogene, and the canine rab7 gene, than to any of the plant ras gene products so far identified (< 40% homology). The high percentage of amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rab, which is the plant counterpart of rab7. Rab/ypt proteins are a subfamily of the ras superfamily thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport. Northern blot hybridisation analysis of total RNA from green and purple podded pea revealed a mRNA species of approximately the same size as the isolated cDNAs.

Rab-GDI presents functional Rab9 to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment.

Dirac-Svejstrup AB, Soldati T, Shapiro AD, Pfeffer SR
J Biol Chem. 1994; 269: 15427-30

Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.

Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae.

Dhar R, Nieto A, Koller R, DeFeo-Jones D, Scolnick EM
Nucleic Acids Res. 1984; 12: 3611-8

A complete nucleotide sequence of two ras-related yeast genes (c- rassc -1 and c- rassc -2) isolated from the yeast strain Saccharomyces cerevisiae is reported. They encode predicted polypeptides of 40,000 and 41,000 daltons, respectively. The N-terminal 170 amino acids from both genes show extensive amino acid homology to other ras genes from vertebrates, whereas their C-termini have diverged. These genes should be useful in the elucidation of a normal biological function of ras-related genes in a simple system like yeast.

Cytoplasmic domain of rhodopsin is essential for post-Golgi vesicle formation in a retinal cell-free system.

Deretic D, Puleo-Scheppke B, Trippe C
J Biol Chem. 1996; 271: 2279-86

In retinal photoreceptors, highly polarized organization of the light-sensitive organelle, the rod outer segment, is maintained by the sorting of rhodopsin and its associated proteins into distinct post-Golgi vesicles that bud from the trans-Golgi network (TGN) and by their vectorial transport toward the rod outer segment. We have developed an assay that reconstitutes the formation of these vesicles in a retinal cell-free system. Vesicle formation in this cell-free assay is ATP-, GTP-, and cytosol-dependent. In frog retinas vesicle budding also proceeds at 0 degrees C, both in vivo and in vitro. Vesicles formed in vitro are indistinguishable from the vesicles formed in vivo by their buoyant density, protein composition, topology, and morphology. In addition to the previously identified G-proteins, these vesicles also contain rab11. Concurrently with vesicle budding, resident proteins are retained in the TGN. Collectively these data suggest that rhodopsin and its associated proteins are sorted upon exit from the TGN in this cell-free system. Removal of membrane-bound GTP-binding proteins of the rab family by rab GDP dissociation inhibitor completely abolishes formation of these vesicles and results in the retention of rhodopsin in the Golgi. A monoclonal antibody to the cytoplasmic (carboxy-terminal) domain of rhodopsin and its Fab fragments strongly inhibit vesicle formation and arrest newly synthesized rhodopsin in the TGN rather than the Golgi. Therefore rhodopsin sorting at the exit from the TGN is mediated by the interaction of its cytoplasmic domain with the intracellular sorting machinery.

Of yeast, mice, and men. Rab proteins and organelle transport.

Deacon SW, Gelfand VI
J Cell Biol. 2001; 152: 214-214

Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex.

Conchon S, Cao X, Barlowe C, Pelham HR
EMBO J. 1999; 18: 3934-46

Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae. Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles. We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3. Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p. Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae. got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes. In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect. Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo. We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi.

Characterization of a Phytophthora infestans gene involved in vesicle transport.

Chen Y, Roxby R
Gene. 1996; 181: 89-94

Members of the Ras superfamily of monomeric GTP-binding proteins have been shown to be essential in specific steps of vesicle transport and secretion in widely divergent organisms. We report here the characterization of a gene from Phytophthora infestans encoding a deduced amino acid (aa) sequence belonging to the Ypt class of monomeric GTP-binding proteins, products shown in other organisms to be essential for vesicle transport between the endoplasmic reticulum and the cis-Golgi compartments. Analysis of genomic and cDNA sequences of this gene, Piypt1, indicates that it contains five introns, one in the 5'-untranslated region. All introns are typical in beginning with GT and ending with AG. The region of the transcription start point displays a number of features characteristic of fungi and other eukaryotes, but it does not contain TATA or CAAT motifs. A single transcript is produced from the gene, which is polyadenylated, but the gene does not contain a recognizable polyadenylation signal. Genomic DNA blots indicate that Piypt1 is a single-copy gene. Comparisons of Ypt1 aa sequences indicate that P. infestans is more closely related to algae and higher plants than to the true fungi. The protein product of the Piypt1 gene, expressed in Escherichia coli, cross-reacts with antiserum against yeast Ypt1 protein and binds GTP. Furthermore, the Piypt1 gene is able to functionally complement a mutant ypt1 gene in Saccharomyces cerevisiae. The aa sequence similarity, immunological cross-reactivity and functional attributes of Piypt1 make it likely that it is an authentic ypt1 gene which participates in vesicle transport in Phytophthora infestans.

The role of ARF and Rab GTPases in membrane transport.

Chavrier P, Goud B
Curr Opin Cell Biol. 1999; 11: 466-75

Two key events of intracellular transport and membrane trafficking in eukaryotic cells, the formation of transport vesicles and their specific delivery to target membranes, are controlled by small GTPases of the ADP-ribosylation factor (ARF) and Rab families, respectively. The past 18 months have seen the identification of proteins that regulate ARF and Rab GDP/GTP cycle, as well as the characterization of their effectors, shedding light on the molecular mechanisms of ARF and Rab function.

The ral gene: a new ras related gene isolated by the use of a synthetic probe.

Chardin P, Tavitian A
EMBO J. 1986; 5: 2203-8

We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B-lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras.

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.

Cao X, Ballew N, Barlowe C
EMBO J. 1998; 17: 2156-65

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.

Rab proteins in gastric parietal cells: evidence for the membrane recycling hypothesis.

Calhoun BC, Goldenring JR
Yale J Biol Med. 1996; 69: 1-8

The gastric parietal cell secretes large quantities of HCl into the lumen of the gastric gland in response to secretagogues such as histamine. In the membrane recycling hypothesis, this secretory activity requires the trafficking of the gastric H+/K(+)-ATPase to the cell surface from intracellular tubulovesicles. The Rab subclass of small GTP-binding proteins is thought to confer specificity to vesicle transport throughout the secretory pathway, and previous investigations established that Rab11 is highly expressed in gastric parietal cells. Recent discoveries in intra-Golgi transport and neuronal synaptic vesicle fusion have fortuitously converged on an evolutionarily conserved protein complex involved in vesicle docking and fusion. Recent results indicate that Rab11 is involved in the apical targeting of vesicles in parietal cells and other epithelial cells throughout the gastrointestinal tract. In support of the membrane recycling hypothesis, Rab co-segregates with H+/K(+)-ATPase in parietal cells. The presence of Rab11 on tubulovesicles supports a role for this Rab protein in recycling vesicle trafficking.

Trimeric G proteins in Golgi transport.

Burgoyne RD
Trends Biochem Sci. 1992; 17: 87-8

Reversal of fortune. Do Rab GTPases act on the target membrane?

Brennwald P
J Cell Biol. 2000; 149: 1-4

Protein transport. A fusion of new ideas.

Bock JB, Scheller RH
Nature. 1997; 387: 133-5

Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in yeast.

Benli M, Doring F, Robinson DG, Yang X, Gallwitz D
EMBO J. 1996; 15: 6460-75

In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.

A gene from Saccharomyces cerevisiae which codes for a protein with significant homology to the bacterial 3-phosphoserine aminotransferase.

Belhumeur P, Fortin N, Clark MW
Yeast. 1994; 10: 385-9

During the sequencing of the gene GSP2 from Saccharomyces cerevisiae, we have encountered an adjacent open reading frame having strong homology to the 3-phosphoserine aminotransferase (E.C.2.6.1.52) from other organisms. In this report, we present the sequence for this yeast SERC, and evidence that its deletion from the yeast genome leads to serine dependency. The sequence has been deposited in the GenBank data library under Accession Number L20917.

How do Rab proteins function in membrane traffic?

Armstrong J
Int J Biochem Cell Biol. 2000; 32: 303-7

The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs. Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p. However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.

Membrane fusion: timing is everything.

Aridor M, Balch WE
Nature. 1996; 383: 220-1

Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes.

Alexandrov K, Horiuchi H, Steele-Mortimer O, Seabra MC, Zerial M
EMBO J. 1994; 13: 5262-73

Rab proteins comprise a family of small GTPases that serve a regulatory role in vesicular membrane traffic. Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their membrane association and function. This post-translational modification is catalysed by rab geranylgeranyl transferase (Rab-GGTase), a multisubunit enzyme consisting of a catalytic heterodimer and an accessory component, named rab escort protein (REP)-1. Previous in vitro studies have suggested that REP-1 presents newly synthesized rab proteins to the catalytic component of the enzyme, and forms a stable complex with the prenylated proteins following the transfer reaction. According to this model, a cellular factor would be required to dissociate the rab protein from REP-1 and to allow it to recycle in the prenylation reaction. RabGDP dissociation inhibitor (RabGDI) was considered an ideal candidate for this role, given its established function in mediating membrane association of prenylated rab proteins. Here we demonstrate that dissociation from REP-1 and binding of rab proteins to the membrane do not require RabGDI or other cytosolic factors. The mechanism of REP-1-mediated membrane association of rab5 appears to be very similar to that mediated by RabGDI. Furthermore, REP-1 and RabGDI share several other functional properties, the ability to inhibit the release of GDP and to remove rab proteins from membranes; however, RabGDI cannot assist in the prenylation reaction. These data suggest that REP-1 is per se sufficient to chaperone newly prenylated rab proteins to their target membranes.