NORMAL GENOMIC

• Nordmann M et al.
• The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7.
• Curr Biol. 2010; 20: 1654-9
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• Rab GTPases coordinate membrane fusion reactions [1]. Rab-GDP requires aguanine nucleotide exchange factor (GEF) for its conversion to the activeGTP form. It then binds to effectors such as multimeric tetheringcomplexes and supports fusion [2]. GTPase-activating proteins (GAPs)promote GTP hydrolysis to inactivate the Rab. GEFs are thus criticalactivators of fusion reactions [3, 4]. The Rab GEF family is diverse,ranging from multimeric complexes [5] to monomeric GEFs [6-9]. At the lateendosome, Rab7 activation is critical for endosomal maturation. The yeastRab7 homolog Ypt7 binds to the homotypic fusion and protein sorting (HOPS)complex [10, 11]. Its subunit Vps39/Vam6 has been proposed as a GEF forYpt7 [12] and the Rag GTPase Gtr1 [13], but other genetic evidence hasimplicated the endosomal protein Ccz1 as a GEF for Ypt7 [14]. Ccz1 and itsbinding partner Mon1 have been linked to endosomal transport andmaturation [15-20]. We now provide evidence that the dimeric Mon1-Ccz1complex is the Rab7/Ypt7 GEF. The Mon1-Ccz1 complex, but neither proteinalone, counteracts GAP function in vivo, rescues in vitro fusion ofvacuoles carrying Ypt7-GDP, and promotes nucleotide exchange on Ypt7independently of Vps39/HOPS. Our data indicate that the Mon1-Ccz1 complextriggers endosomal maturation by activating Ypt7 on late endosomes.

• Mattera R, Tsai YC, Weissman AM, Bonifacino JS
• The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub)and functions as a Ub ligase through an atypical Ub-interacting motif anda zinc finger domain.
• J Biol Chem. 2006; 281: 6874-83
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• Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotideexchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, amultivalent adaptor protein that also binds to Rab4, Rab5, and to domainspresent in gamma-adaptins and the Golgi-localized, gamma-ear-containing,ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchangeactivity, thus generating GTP-bound, membrane-associated Rab5 that, inturn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex onendosomes. Although the Rabex-5.Rabaptin-5 complex is critical to theregulation of endosomal fusion, the structural determinants of thisinteraction are unknown. Likewise, the possible binding and covalentattachment of ubiquitin to Rabex-5, two modifications that are critical tothe function of yeast Vps9p in endosomal transport, have not been studied.In this study, we identify the 401-462 and 551-661 coiled-coils as theregions in Rabex-5 and Rabaptin-5, respectively, that interact with oneanother. We also demonstrate that Rabex-5 undergoes ubiquitination andbinds ubiquitin, though not via its proposed C-terminal CUE-like domain.Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising anA20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is importantfor ubiquitin binding and ubiquitination. Importantly, we demonstrate thatthe Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. Theseobservations extend our understanding of the regulation of Rabex-5 byRabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligasethat binds ubiquitin and undergoes ubiquitination indicates that its rolein endosome fusion may be subject to additional regulation byubiquitin-dependent modifications.

• Prescianotto-Baschong C, Riezman H
• Ordering of compartments in the yeast endocytic pathway.
• Traffic. 2002; 3: 37-49
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• We have characterized the morphology of the yeast endocytic pathwayleading from the plasma membrane to the vacuole by following thetrafficking of positively charged nanogold in combination with compartmentidentification using immunolocalization of t-SNARE proteins. The firstendocytic compartment, termed the early/recycling endosome, contains thet-SNARE, Tlg1p. The next compartment, the prevacuolar compartment,contains Pep12p. After transport to the prevacuolar compartment, wherevacuolar enzymes are seen on their way to the vacuole, endocytic contentis delivered to the late endosome and on to the vacuole, both of which aredevoid of Pep12p immunolabel. Traffic to the prevacuolar compartment isreduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, andYpt53p and in vps27 mutant cells. On the other hand, traffic to the earlyrecycling endosome is less dependent on Rab5 homologs and does not requireVps27p.

• Sjolinder M, Uhlmann J, Ponstingl H
• DelGEF, a homologue of the Ran guanine nucleotide exchange factor RanGEF,binds to the exocyst component Sec5 and modulates secretion.
• FEBS Lett. 2002; 532: 211-5
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• In order to identify the function of deafness locus putative guaninenucleotide exchange factor (DelGEF), a protein homologous to thenucleotide exchange factor for the small GTPase Ran, a cDNA library wasscreened for interacting proteins using a yeast two-hybrid system. Thehuman homologue of Sec5, a protein involved in vesicle transport andsecretion, was identified as a binding partner. The interaction betweenDelGEF and Sec5 was found to be dependent on Mg2+ and stimulated byguanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP).Downregulation of endogenous DelGEF in HeLa cells induced increasedextracellular secretion of proteoglycans indicating a possible role forDelGEF in the secretion process.

• Strick DJ, Francescutti DM, Zhao Y, Elferink LA
• Mammalian suppressor of Sec4 modulates the inhibitory effect of Rab15during early endocytosis.
• J Biol Chem. 2002; 277: 32722-9
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• Rab15 is a novel endocytic Rab that counters the stimulatory effect ofRab5-GTP on early endocytic trafficking. Rab15 may interfere with Rab5function directly by sequestering Rab5 effectors or indirectly throughnovel sets of effector interactions. To distinguish between thesepossibilities, we examined the effector binding properties of Rab15. Rab15does not interact directly with the Rab5 effectors rabex-5 and rabaptin-5in a yeast two-hybrid binding assay. Rather mammalian suppressor of Sec4(Mss4) was identified as a binding partner for Rab15. Mss4 preferentiallybinds GDP-bound (T22N) and nucleotide-free (N121I) Rab15, consistent withthe proposed role of Mss4 as a chaperone that stabilizes target Rabs intheir nucleotide-free form. Mutational analysis of Rab15 indicates thatlysine at position 48 (K48Q) is important for the binding of Rab15-GDP toMss4. Moreover, the mutation K48Q counters the inhibitory phenotype ofwild type Rab15 on receptor-mediated endocytosis in HeLa cells andhomotypic endosome fusion in vitro without altering the relative amount ofcell surface-associated transferrin receptor. Together, these dataindicate a novel role for Mss4 as an effector for Rab15 in early endocytictrafficking.

• Finken-Eigen M, Muller S, Kohrer K
• Cloning and characterization of a dominant-negative vps1 allele of theyeast Saccharomyces cerevisiae.
• Biol Chem. 1997; 378: 1187-91
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• The gene product of the yeast VPS1 gene is a member of a family ofhigh-molecular-weight GTP-binding proteins that are involved in diversecellular processes. The Vps1 protein (Vps1p) was shown to perform anessential function in the yeast secretory pathway. Here, we report theisolation and characterization of a mutant allele of the VPS1 gene,causing a dominant-negative vacuolar protein sorting (vps) defect, asdemonstrated by the mislocalization of the vacuolar hydrolasecarboxypeptidase Y (CPY). DNA sequence analysis of the mutant vps1 allele(vps1d-293) revealed a single point mutation, resulting in an amino acidexchange at position 293 from Ala to Asp. The mutation is locateddownstream of the tripartite GTP-binding motif found in the amino-terminalhalf of the protein. The observation that expression of wild-type Vps1ppartially suppressed the dominant-negative CPY sorting phenotype indicatescompetition of a non-functional mutant Vps1 protein and a functionalwild-type VPS1p for a Vps1p-binding site of an as yet unknown vacuolarprotein sorting factor.

• Garrett MD, Zahner JE, Cheney CM, Novick PJ
• GDI1 encodes a GDP dissociation inhibitor that plays an essential role inthe yeast secretory pathway.
• EMBO J. 1994; 13: 1718-28
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• GTP binding proteins of the Sec4/Ypt/rab family regulate distinctvesicular traffic events in eukaryotic cells. We have cloned GDI1, anessential homolog of bovine rab GDI (GDP dissociation inhibitor) from theyeast Saccharomyces cerevisiae. Analogous to the bovine protein, purifiedGdi1p slows the dissociation of GDP from Sec4p and releases the GDP-boundform from yeast membranes. Depletion of Gdi1p in vivo leads to loss of thesoluble pool of Sec4p and inhibition of protein transport at multiplestages of the secretory pathway. Complementation analysis indicates thatGDI1 is allelic to sec19-1. These results establish that Gdi1p plays anessential function in membrane traffic and are consistent with a role forGdi1p in the recycling of proteins of the Sec4/Ypt/rab family from theirtarget membranes back to their vesicular pools.

• Ekena K, Vater CA, Raymond CK, Stevens TH
• The VPS1 protein is a dynamin-like GTPase required for sorting proteins tothe yeast vacuole.
• Ciba Found Symp. 1993; 176: 198-211
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• VPS1 encodes a 79 kDa protein required for the proper sorting of solublevacuolar proteins in Saccharomyces cerevisiae. The N-terminal half ofVps1p, which contains a consensus GTP-binding motif, shares extensivehomology with a growing family of high molecular mass GTP-bindingproteins. Members of this family have been implicated in a number ofcellular processes. Vps1p most closely resembles themicrotubule-associated protein dynamin. As predicted from the sequence,Vps1p binds and hydrolyses GTP. However, no requirement for microtubuleswas found for Vps1p function in protein sorting. In subcellularfractionation experiments Vps1p associates with the membrane fraction; theC-terminal half of Vps1p is important for this association. Mutationalanalysis of VPS1 generated two classes of mutations, dominant negative andrecessive. The dominant mutations all mapped to the N-terminal half of theprotein. Recessive mutations gave rise to either truncated or unstableproteins. A potential Vps1p-interacting protein (Mvp1p) has been isolatedby screening for suppressors of the dominant alleles of VPS1. Takentogether these results suggest that Vps1p is a two-domain protein that ispart of a multi-subunit protein complex involved in vacuolar proteinsorting.