Small GTPases 2:3, 182-186; May/June 2011; © 2011 Landes Bioscience

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182 Small GTPases volume 2 issue 3

Extra View to: Nordmann M, Cabrera M, Perz A, Bröcker C, Ostrowicz C, Engelbrecht-Vandré S, et al. The Mon1- Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr Biol 2010; 20:1654–9; PMID: 20797862; DOI: 10.1016/j.cub.2010.08.002.andOstrowicz CW, Bröcker C, Ahnert F, Nordmann M, Lachmann J, Peplowska K, et al. Defined subunit arrangement and rab interactions are required for functionality of the HOPS tethering complex. Traffic 2010; 11:1334–46; PMID: 20604902; DOI: 10.1111/ j.1600-0854.2010.01097.x.

Key words: membrane fusion, Rab GTPases, tethers, SNAREs, HOPS, CORVET, Ypt7, Vps21, Mon1-Ccz1

Submitted: 05/07/11

Revised: 05/24/11

Accepted: 05/30/11

DOI: 10.4161/sgtp.2.3.16701

*Correspondence to: Siegfried Engelbrecht-Vandré; Email: engel@uos.de

within eukaryotic cells, rab Gtpases control the maturation

of early to late endosomes and their sub-sequent fusion with the vacuole. within this Extraview, we will focus on our recent findings regarding the activation of the rab7 homolog ypt7 in yeast and its interplay with the two multisubunit tethering complexes corvEt and hops.

Introduction

Eukaryotes are distinguished, among other features, by their requirement and ability to maintain subcellular com-partments. This relies on continuous, import-export balanced supply of lipids and proteins. Protein and lipid trafficking occur by budding and scission of transport vesicles at one organelle, directed or dif-fusion driven transport to the destination, and finally fusion of transport vesicles with the target membrane. These events are precisely controlled in time and space by a variety of proteins and lipids. Among these are small GTPases along with their regulators and effectors, adaptor proteins, coat proteins, tethers, SNAREs, and a variety of phosphoinositol derivatives.

Here, we focus on events in the yeast endocytic pathway. Endocytic vesicles transport ubiquitinated receptors from the plasma membrane to early endosomes, which are transformed to late endosomes/multivesicular bodies to finally fuse with the lysosome or vacuole. The process is critical for the turn-over and recycling of the plasma membrane constituents, and ubiquitin is known as a membrane iden-tifier of the maturing endosome. This transport route is not merely unidirec-tional, as it also provides means to recycle

rab Gtpases and tethering in the yeast endocytic pathway

Jens Lachmann, Christian Ungermann and Siegfried Engelbrecht-Vandré*Department of Biology/Chemistry; Biochemistry Section; University of Osnabrück; Osnabrück, Germany

for instance endocytosed receptors back to the plasma membrane, which otherwise would be digested in the vacuole. On its way, the endosomal compartment merges with Golgi-derived vesicles of the biosyn-thetic pathway, which deliver hydrolases to the vacuole. Another pathway origi-nates directly at the trans Golgi network, and is called the AP-3 pathway because of its special adaptor protein complex. It carries specific membrane proteins, which thus avoid the multivesicular bodies and the associated sorting machinery into the vacuole lumen. In addition, homotypic vacuole-vacuole fusion takes place, pri-marily after cytokinesis.

Vesicle fusion with a target membrane in general consists of reversible first con-tacts provided by a small Rab GTPase located on the vesicle and its effector— a tether protein, which is recruited from the cytosol. Tethers are either coiled-coil proteins like Vac1/EEA1, acting at the early endosome, or multisubunit tether-ing complexes (MTCs) with up to ten subunits like the heterohexameric HOPS complex.1 They bridge opposing mem-branes as direct effectors of Rab GTPases, and interact with lipids and SNAREs. The SNAREs are present on opposing mem-branes, form specific intermolecular four helix bundles during membrane docking, and thus catalyze membrane fusion.2 The subsequently formed SNARE complexes on the fused membranes are thereafter disassembled by the AAA-ATPase Sec18/NSF and its adaptor protein Sec17/a-SNAP. SNAREs can thus be reused after fusion.

MTCs are thought to specifically medi-ate the SNARE assembly and proofread the assembled complex. At least in yeast, the HOPS tethering complex was shown

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membrane by shielding its hydrophobic prenyl anchor. Reinsertion into the mem-brane for another round of activation may be assisted then by a GDI dissociation fac-tor (GDF).9 The C-terminal Cys motif of Rab GTPases is preceded by a hypervari-able region of 35–40 residues, also essen-tial for localization, membrane binding and interaction with GDI and REP.10

Maturing organelles pose a problem in identification, since they gradually change in surface composition. An inter-esting model explains this by a linear arrangement of Rab GTPases.2,5,11,12 In a way, such a series resembles a counter cur-rent distribution, in that one active Rab both recruits the GEF for the follow-ing Rab, which thereby is turned on and at the same time binds the GAP for the preceding Rab, which thereby is switched off.5,13,14

Regulation of Rab GTPases in Yeast Endocytosis

In yeast, early endosomes contain the Rab5-like Vps21, whereas late endosomes and the vacuole contain the Rab7 homolog Ypt7. It is assumed that the distribution of these two both reflects and determines organelle identity. figure 1 summarizes

to prevent premature disassembly of the SNAREs.3 Thus, tethering complexes are multifunctional, since they can interact with both a small GTPase and SNAREs. Whether this occurs sequentially or con-comitantly and who comes first is a most interesting open question.1,3,4

Among the five principal families of small GTPases the Rabs [“Ras” = (Rat sarcoma)-related in brain] are central regulators of vesicle budding, motility, and fusion. They also comprise the larg-est number: 11 in S. cerevisiae, > 60 in humans, and close to 60 in Arabidopsis thaliana.5 This factor of ~5 reflects the more demanding requirements both in terms of specialization and diversification in higher eukaryotes as compared with yeast.

Rab GTPases cycle between a GTP- and a GDP-bound state. The difference is most evident in two so-called switch regions, which are much more ordered if GTP is bound to the Rab. Only in the active GTP-form the Rab interacts with its effector. Despite their name, small GTPases are poor catalysts, their intrin-sic hydrolysis rate is very low. Hydrolysis is accelerated by binding of a GTPase activating protein (GAP), which harbors the well-conserved and indispensable

TBC (Tre-2/Bub2/Cdc16) domain. This domain encodes an “arginine finger” and a “glutamine finger”, which both comple-ment the active site and stabilize the tran-sition state for GTP-hydrolysis. The cycle of the GTPase is completed by a guanine nucleotide exchange factor (GEF) that catalyzes the quick exchange from GDP to GTP, which is at least 10-fold more abun-dant in cells than GDP. GTPases thus require both a GAP and a GEF in order to be switchable between “on” and “off”. 5-7

Organelles of the endomembrane sys-tem are distinguished from each other functionally and hence structurally: both their limiting membranes may vary in lipid and protein composition, includ-ing Rab GTPases. This raises the ques-tion about the Rab localization. Rab GTPases are posttranslationally modified at their C-termini by geranylgeranylation, which serves as a hydrophobic membrane anchor. Upon their synthesis, they bind GDP and then attach to the Rab escort protein (REP), followed by prenyl tail addition through geranylgeranyl trans-ferase (RabGGT), and delivery to the target membrane.8 There, the GEF-GAP cycle takes place. After inactivation, a guanine nucleotide dissociation inhibitor (GDI) can extract the GDP-Rab from the

Figure 1. Structural data on the two rab GTPases vps21 (Ypt51) and Ypt7 from S. cerevisiae. (a) secondary structure assignment and sequence align-ment, (B) overlay of 3d structures. The assignments in (a) (labeled “dssp”) and the overlay in (B) were made with YaSara 15 based on pdb entries 1ky2 and 1ek0 (www.pdb.org). The crystal structures describe residues 5–174 of vps21 and 3–182 of Ypt7, the hypervariable domains were removed. “Switch 1” and “Switch 2” indicate the regions that change conformation most dramatically upon binding of GTP. The hypervariable domain is important in targeting and for interaction with rEP and GDi (compare text). C = coil, E = b strand, H = a helix, t = turn, 3 = 310 helix. The alignment was made with TCoffee.16 in B vps21 is shown in red and the bound GTP analog in yellow, Ypt7/GPPNHP in light blue/green. The N- and C-termini are colored blue, they are barely visible in the lower portion and at the back of the molecules. The hypervariable domains thus also would extend away from the viewer. The two switch regions are colored blue for Ypt7, Switch 1 is at the left border and Switch 2 at the lower left.

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game, namely GEF, GDI, Rab, GDP, and GTP.25-27 figure 2 presents a model con-sistent with our current knowledge of the events at the maturing endosome.

Rab-Mediated Tethering in Yeast Endocytosis

As discussed, GTP-loaded Rabs bind their effectors on membranes and pro-mote membrane remodelling and the subsequent fusion. On early endosomes Vps21-GTP interacts with the tethering complex CORVET (class C core vacuole/endosome tethering), and at the late endo-some and vacuole, Ypt7-GTP interacts with the homologous HOPS (homotypic fusion and protein sorting) complex.28-30 These two heterohexameric complexes share four out of six subunits, the class C core subunits (Vps 11, 16, 18, 33), and two complex-specific subunits (CORVET: Vps3 and 8; HOPS: Vps39 and 41). Both interact with their specific Rab. Within the CORVET complex, Vps3 and Vps8 bind to Vps21-GTP.28,31,32 HOPS binds to Ypt7-GTP via its subunit Vps41.33,34 The HOPS specific subunit Vps39 also

Does the activated Ypt7 also sequester the GAP for the preceding Rab, Vps21, thereby completing the cascade? In C. elegans Mon1 can shut down the previ-ous GEF and TBC-2 has been identified as a GAP for RAB-5, that needs RAB-7 to localize on the late endosome.22,23 Mammalian RabGAP-5 specifically blocks the EGF uptake in early endocy-tosis by deactivating Rab5.24 In yeast, no specific GAP for Vps21 has been identified so far. So at least a portion of the afore-mentioned chain of events (an active Rab recruits the GEF for the following Rab) seems to occur during the transition from early to late endosomes (table 1) also in yeast.

It remains unclear whether endosomal Rabs really require a GDF to be displaced from GDI, when recruited to membranes. With respect to DrrA from Legionella, the GEF activity of this protein in vitro is sufficient to displace GDI and con-comitantly convert Rab1 to the GTP form. This can be explained on thermo-dynamic grounds and requires an appro-priate match between concentrations and affinities of and between the players in the

some structural features of these two Rab GTPases.

During endocytosis of ubiquitinated plasma membrane receptors, Vps21 is acti-vated by its GEF Vps9 (Rabex5 in mam-mals), and promotes fusion with early endosomes.17,18 As these mature by fusion with incoming Golgi-derived vesicles and upon formation of intralumenal vesicles (ILVs), Ypt7 is recruited to the late endo-some. ILVs in the maturing MVBs contain receptors that are cleared of ubiquitin dur-ing their sorting. This in turn also changes the surface composition of endosomes. We demonstrated that Mon1-Ccz1 is the GEF for Ypt7, and that it requires Vps21 and its effector, the CORVET complex, to bind to endosomes.19 These data are supported by recent findings from other organisms. In C. elegans, the Mon1 homolog SAND-1 is sufficient to displace Rabex5 from endo-somes. This interrupts the permanent activation of Rab5 and promotes binding of Rab7 to late endosomes.20 The mam-malian Mon1 interacts with Rab5-GTP, but only the complex of Mon1-Ccz1 binds Rab7, and seems to promote its activation and release from GDI.21

Figure 2. working model of the transition from the early to the late endosome/MvB (modified from Nordmann et al.19). The activation of vps21 takes place at the plasma membrane or endocytic vesicle via vps9. at the early endosome, vps21 recruits its effectors, vps3 and vps8, and therefore the COrvET complex. This module is then required for the recruitment of the Mon1-Ccz1 complex, which catalyzes the nucleotide exchange of the down-stream rab GTPase Ypt7, and hence activates its signaling. it is not known so far, if the targeting of Ypt7 is supported by a GDF or possibly by a vps21 effector. Once activated, Ypt7 binds the HOPS complex for the fusion of the MvB with the vacuole. whether the COrvET either matures into the HOPS, or both are recruited independently, remains to be clarified. Likewise, a specific vps21 GaP is not known. The activation state of the rabs is depicted by a spiny (GDP) or a smooth shape (GTP). Double-headed arrows indicate likely interactions, question marks point out gaps in our knowledge. The size of the protein icons does not reflect their respective masses.

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complexes, may influence the formation of CORVET and HOPS.32,34 Independently, Vps41–39 and Vps8–3 subcomplexes have been reported.32 Although we could not recover these complexes from yeast so far, their existence suggests that HOPS and CORVET may assemble in the cytosol. It thus remains an open question whether CORVET is rebuilt into HOPS (the opposite would appear rather improb-able in view of the chronological order of events), or whether both complexes are assembled independently. The existence of the hybrid hexamer Vps41-Vps3-C core certainly points into the former direction, with the higher affinity subunit (Vps39) taking over from the lower affinity (Vps3) subunit. A hybrid variant Vps8-Vps39-C core may also exist. In our model, it would possibly strongly interfere with a unidirectional transition from CORVET into HOPS. In any case, the concomi-tant presence of all six subunits in the cytosol would require some sort of usher to allow for incorporation of the lower affinity subunits in the first place. The two Rabs, Vps21 and Ypt7 would come in handy for this purpose then, perhaps serving as nucleation points for the follow-ing steps. How the exchange of Rabs and maturation/change of tethers is linked to endosomal maturation is not yet clear. It is possible that the reduction of ubiq-uitinated receptors on the organelle con-tributes to this transition. The existence of significant amounts of both Ypt7 and HOPS already at the late endosome would call for a hand-over from Vps21-bound CORVET. At the extreme, CORVET could also be a mere precursor of the ulti-mately important HOPS, perhaps even devoid of tethering activity by itself.

Usually, one would expect a tethering complex to reside on the target membrane, not on an incoming transport vesicle. In view of the SNARE binding subunit Vps33, a binding via this fixation also is possible, especially since the number of Sec1/Munc18 like proteins in yeast (four) is much smaller than the number of char-acterized SNARE complexes (eight), thus calling for redundancy in binding. The finding that HOPS prevents premature disassembly of preassembled SNAREs also perfectly fits the picture.3 In addi-tion, HOPS binds the vacuolar SNARE

recognizes Ypt7, but apparently indepen-dently of its nucleotide load and only as monomeric, free Vps39, a rather puzzling finding.29,32,34 It was recently proposed that Vps3 and Vps8, as well as Vps41 and Vps39 build up an interaction module to recognize their appropriate Rabs.32 This could explain the different behavior of monomeric Vps39 in vitro.

Deletion of each of the class C core subunits leads to a complete fragmenta-tion of the vacuole, consistent with their central function within two complexes.35 Strikingly, five of the six subunits in each complex have the same predicted domain structure, a N-terminal portion likely forming a b-propeller, followed by a putative a-solenoid domain.34,36 The one exception is Vps33, which bears strong homology to SNARE-interacting Sec1/Munc18-like proteins.37 To obtain some insight into the assembly of the two com-plexes, we recently used a deletion and overexpression approach. We could show that the Rab-specific subunits Vps41 and Vps39 occupy one pole of the HOPS com-plex, whereas the SNARE specific sub-unit Vps33 is located at the opposite one (fig. 2).34 In view of the identical core subunits and pronounced structural simi-larities between the other four subunits, a similar arrangement is likely for the CORVET complex. Independent yeast-2 hybrid analyses confirmed these observa-tions.32 Importantly, the two complexes have different fates if single subunits are deleted. The loss of CORVET Vps3 left a pentamer behind, whereas the deletion of Vps8 resulted in a hybrid hexamer, con-sisting of Vps3, Vps41 and the four core subunits. A similar hybrid was obtained in vps39D cells.28,34

The subunit composition of both complexes suggested early on that they could originate from a common core, which would then be completed to form either CORVET or HOPS. However, we never obtained a tetramer of the core subunits, but rather a dimeric Vps16-Vps33 subcomplex, which also turned up if any central core subunit (Vps11 or 18) was deleted. In the same mutant back-ground, a subcomplex of the class C core subunit Vps11 with Vps39 or CORVET Vps3 was observed. The C-terminal part of Vps11, likely the central hub for both

complex in vitro.38 At the late endosome/vacuole, just as in homotypic fusion of two vacuoles, the situation, however, is special in that both Rab (Ypt7) and tether (HOPS) are present on both participating membranes. Therefore, a clear-cut deci-sion between these possibilities awaits fur-ther experimentation.

As mentioned above, the endocytic pathway provides means to recycle cer-tain compounds rather than degrade them ultimately in the vacuole. An essential part of this process is the retromer complex, which consists of a cargo-selective and a membrane-tubulating subcomplex.39 The cargo-selective subcomplex (Vps26–29–35) binds to Ypt7-GTP such that the Rab GTPase contributes to the targeting and recruitment of the retromer to endosomal membranes.40-42 Since Ypt7 is not exclu-sively located at the vacuole, but turns up earlier at late endosomes, this finding calls for an integrative role for Ypt7, balancing recycling and degradative processes. Thus, Rab GTPases (and other small GTPases just as well) not only cycle between ‘on’ and ‘off ’, and membrane bound and cyto-solic states, they also take part in cellular networking by a plethora of downstream effectors and, likely, regulators.

acknowledgments

Work in the authors’ laboratory was sup-ported by the DFG (grant UN111/5–2, SFB 944) and the Hans-Mühlenhoff foundation (to C.U.).

references1. Bröcker C, Engelbrecht-Vandré S, Ungermann C.

Multisubunit tethering complexes and their role in membrane fusion. Curr Biol 2010; 20:R943-52; PMID: 21056839; DOI:10.1016/j.cub.2010.09.015.

2. Cai H, Reinisch K, Ferro-Novick S. Coats, Tethers, Rabs, and SNAREs Work Together to Mediate the Intracellular Destination of a Transport Vesicle. Dev Cell 2007; 12:671-82; PMID: 17488620; DOI:10.1016/j.devcel.2007.04.005.

3. Hickey CM, Wickner W. HOPS initiates vacu-ole docking by tethering membranes before trans-SNARE complex assembly. Mol Biol Cell 2010; 21:2297-305; PMID: 20462954; DOI:10.1091/mbc.E10-01-0044.

4. Wickner W. Membrane Fusion: Five Lipids, Four SNAREs, Three Chaperones, Two Nucleotides, and a Rab, All Dancing in a Ring on Yeast Vacuoles. Annu Rev Cell Dev Biol 2010; 26:115-36; PMID: 20521906; DOI:10.1146/annurev-cell-bio-100109-104131.

5. Hutagalung AH, Novick PJ. Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 2011; 91:119-49; PMID: 21248164; DOI:10.1152/physrev.00059.2009.

186 Small GTPases volume 2 issue 3

32. Plemel RL, Lobingier BT, Brett CL, Angers CG, Nickerson DP, Paulsel A, et al. Subunit organiza-tion and Rab interactions of Vps-C protein com-plexes that control endolysosomal membrane traffic. Mol Biol Cell 2011; 22:1353-63; PMID: 21325627; DOI:10.1091/mbc.E10-03-0260.

33. Brett CL, Plemel RL, Lobinger BT, Vignali M, Fields S, Merz AJ. Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase. J Cell Biol 2008; 182:1141-51; PMID: 18809726; DOI:10.1083/jcb.200801001.

34. Ostrowicz CW, Bröcker C, Ahnert F, Nordmann M, Lachmann J, Peplowska K, et al. Defined subunit arrangement and rab interactions are required for functionality of the HOPS tethering complex. Traffic 2010; 11:1334-46; PMID: 20604902; DOI:10.1111/j.1600-0854.2010.01097.x.

35. Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 1992; 3:1389-402; PMID: 1493335.

36. Nickerson DP, Brett CL, Merz AJ. Vps-C com-plexes: gatekeepers of endolysosomal traffic. Curr Opin Cell Biol 2009; 21:543-51; PMID: 19577915; DOI:10.1016/j.ceb.2009.05.007.

37. Südhof TC, Rothman JE. Membrane fusion: grap-pling with SNARE and SM proteins. Science 2009; 323:474-7; PMID: 19164740; DOI:10.1126/sci-ence.1161748.

38. Krämer L, Ungermann C. HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites. MBoC in Press 2011; DOI:10.1091/mbc.E11-02-0104.

39. Bonifacino JS, Hurley JH. Retromer. Curr Opin Cell Biol 2008; 20:427-36; PMID: 18472259; DOI:10.1016/j.ceb.2008.03.009.

40. Balderhaar HJK, Arlt H, Ostrowicz C, Bröcker C, Sündermann F, Brandt R, et al. The Rab GTPase Ypt7 is linked to retromer-mediated receptor recy-cling and fusion at the yeast late endosome. J Cell Sci 2010; 123:4085-94; PMID: 21062894; DOI:10.1242/jcs.071977.

41. Seaman MNJ, Harbour ME, Tattersall D, Read E, Bright N. Membrane recruitment of the cargo-selective retromer subcomplex is catalysed by the small GTPase Rab7 and inhibited by the Rab-GAP TBC1D5. J Cell Sci 2009; 122:2371-82; PMID: 19531583; DOI:10.1242/jcs.048686.

42. Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol 2006; 7:568-79; PMID: 16936697; DOI:10.1038/nrm1985.

20. Poteryaev D, Datta S, Ackema K, Zerial M, Spang A. Identification of the switch in early-to-late endo-some transition. Cell 2010; 141:497-508; PMID: 20434987; DOI:10.1016/j.cell.2010.03.011.

21. Kinchen JM, Ravichandran KS. Identification of two evolutionarily conserved genes regulating processing of engulfed apoptotic cells. Nature 2010; 464:778-82; PMID: 20305638; DOI:10.1038/nature08853.

22. Li W, Zou W, Zhao D, Yan J, Zhu Z, Lu J, et al. C. elegans Rab GTPase activating protein TBC-2 pro-motes cell corpse degradation by regulating the small GTPase RAB-5. Development 2009; 136:2445-55; PMID: 19542357; DOI:10.1242/dev.035949.

23. Chotard L, Mishra AK, Sylvain M, Tuck S, Lambright DG, Rocheleau CE. TBC-2 regulates RAB-5/RAB-7-mediated endosomal trafficking in Caenorhabditis elegans. Mol Biol Cell 2010; 21:2285-96; PMID: 20462958; DOI:10.1091/mbc.E09-11-0947.

24. Haas AK, Fuchs E, Kopajtich R, Barr FA. A GTPase-activating protein controls Rab5 function in endocyt-ic trafficking. Nat Cell Biol 2005; 7:887-93; PMID: 16086013; DOI:10.1038/ncb1290.

25. Suh HY, Lee D, Lee K, Ku B, Choi S, Woo J, et al. Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA. EMBO J 2010; 29:496-504; PMID: 19942850; DOI:10.1038/emboj.2009.347.

26. Zhu Y, Hu L, Zhou Y, Yao Q, Liu L, Shao F. Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA. Proc Natl Acad Sci USA 2010; 107:4699-704; PMID: 20176951; DOI:10.1073/pnas.0914231107.

27. Schoebel S, Oesterlin LK, Blankenfeldt W, Goody RS, Itzen A. RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleo-tide exchange activity. Mol Cell 2009; 36:1060-72; PMID: 20064470; DOI:10.1016/j.mol-cel.2009.11.014.

28. Peplowska K, Markgraf DF, Ostrowicz CW, Bange G, Ungermann C. The CORVET tethering complex interacts with the yeast Rab5 homolog Vps21 and is involved in endo-lysosomal biogenesis. Dev Cell 2007; 12:739-50; PMID: 17488625; DOI:10.1016/j.devcel.2007.03.006.

29. Wurmser AE, Sato TK, Emr SD. New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J Cell Biol 2000; 151:551-62; PMID: 11062257; DOI:10.1083/jcb.151.3.551.

30. Seals DF, Eitzen G, Margolis N, Wickner WT, Price AA. Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion. Proc Natl Acad Sci USA 2000; 97:9402-7; PMID: 10944212; DOI:10.1073/pnas.97.17.9402.

31. Markgraf DF, Ahnert F, Arlt H, Mari M, Peplowska K, Epp N, et al. The CORVET subunit Vps8 cooper-ates with the Rab5 homolog Vps21 to induce cluster-ing of late endosomal compartments. Mol Biol Cell 2009; 20:5276-89; PMID: 19828734; DOI:10.1091/mbc.E09-06-0521.

6. Barr F, Lambright DG. Rab GEFs and GAPs. Curr Opin Cell Biol 2010; 22:461-70; PMID: 20466531; DOI:10.1016/j.ceb.2010.04.007.

7. Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 2009; 10:513-25; PMID: 19603039; DOI:10.1038/nrm2728.

8. Pereira-Leal JB, Hume AN, Seabra MC. Prenylation of Rab GTPases: molecular mechanisms and involve-ment in genetic disease. FEBS Lett 2001; 498:197-200; PMID: 11412856; DOI:10.1016/S0014-5793(01)02483-8.

9. Pfeffer S, Aivazian D. Targeting Rab GTPases to distinct membrane compartments. Nat Rev Mol Cell Biol 2004; 5:886-96; PMID: 15520808; DOI:10.1038/nrm1500.

10. Pfeffer SR. Structural clues to Rab GTPase func-tional diversity. J Biol Chem 2005; 280:15485-8; PMID: 15746102; DOI:10.1074/jbc.R500003200.

11. Novick P, Medkova M, Dong G, Hutagalung A, Reinisch K, Grosshans B. Interactions between Rabs, tethers, SNAREs and their regulators in exocyto-sis. Biochem Soc Trans 2006; 34:683-6; PMID: 17052174; DOI:10.1042/BST0340683.

12. Rivera-Molina FE, Novick PJ. A Rab GAP cascade defines the boundary between two Rab GTPases on the secretory pathway. Proc Natl Acad Sci USA 2009; 106:14408-13; PMID: 19666511; DOI:10.1073/pnas.0906536106.

13. Del Conte-Zerial P, Brusch L, Rink JC, Collinet C, Kalaidzidis Y, Zerial M, et al. Membrane identity and GTPase cascades regulated by toggle and cut-out switches. Mol Syst Biol 2008; 4:206; PMID: 18628746; DOI:10.1038/msb.2008.45.

14. Rink J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 2005; 122:735-49; PMID: 16143105; DOI:10.1016/j.cell.2005.06.043.

15. Krieger E, Koraimann G, Vriend G. Increasing the precision of comparative models with YASARA NOVA-a self-parameterizing force field. Proteins 2002; 47:393-402; PMID: 11948792; DOI:10.1002/prot.10104.

16. Notredame C. T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000; 302:205-17; PMID: 10964570; DOI:10.1006/jmbi.2000.4042.

17. Carney DS, Davies BA, Horazdovsky BF. Vps9 domain-containing proteins: activators of Rab5 GTPases from yeast to neurons. Trends Cell Biol 2006; 16:27-35; PMID: 16330212; DOI:10.1016/j.tcb.2005.11.001.

18. Horiuchi H, Lippé R, McBride HM, Rubino M, Woodman P, Stenmark H, et al. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 1997; 90:1149-59; PMID: 9323142; DOI:10.1016/S0092-8674(00)80380-3.

19. Nordmann M, Cabrera M, Perz A, Bröcker C, Ostrowicz C, Engelbrecht-Vandré S, et al. The Mon1-Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr Biol 2010; 20:1654-9; PMID: 20797862; DOI:10.1016/j.cub.2010.08.002.