Protein Transport from the Late Golgi to the Vacuole in the Yeast Saccharomyces Cerevisiae

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Biochimica et Biophysica Acta 1744 (2005) 438 – 454 http://www.elsevier.com/locate/bba Review Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae

Katherine Bowersa, Tom H. Stevensb,*

aCambridge Institute for Medical Research and Department of Clinical, Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, UK bInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA

Received 16 March 2005; received in revised form 15 April 2005; accepted 19 April 2005 Available online 11 May 2005

Abstract

The late Golgi compartment is a major protein sorting station in the cell. Secreted proteins, cell surface proteins, and proteins destined for endosomes or lysosomes must be sorted from one another at this compartment and targeted to their correct destinations. The molecular details of protein trafficking pathways from the late Golgi to the endosomal system are becoming increasingly well understood due in part to information obtained by genetic analysis of yeast. It is now clear that proteins identified in yeast have functional homologues (orthologues) in higher organisms. We will review the molecular mechanisms of protein targeting from the late Golgi to endosomes and to the vacuole (the equivalent of the mammalian lysosome) of the budding yeast Saccharomyces cerevisiae. D 2005 Elsevier B.V. All rights reserved.

Keywords: Vacuole protein sorting (VPS); Late Golgi; Multivesicular body (MVB); Endosome; Protein trafficking; Yeast

1. Introduction are defective for vacuolar segregation, and grd mutants are Golgi retention deficient [1–10]. There is significant One essential feature of eukaryotic cells is the presence overlap of the genes identified in these screens. For the of intracellular membrane-bound compartments or organ- purposes of this review, we will focus on the function of elles. Proteins must be targeted to the correct organelle for the VPS genes, and the proteins they encode (see their function, modification, and/or destruction. Genetic Table 1). analysis in yeast has been instrumental in determining the As can be seen from Fig. 1, there are several transport molecular details of protein trafficking pathways and over pathways from the yeast late Golgi compartment. There are 70 proteins involved in the steps of protein trafficking at least two types of vesicles that take proteins to the cell from the yeast late Golgi to the vacuole have now been surface for secretion from the cell or incorporation into the identified. Several large-scale screens have been employed plasma membrane: one is likely to be a direct route, and to find mutants defective in this process. For example, vps another via endosomes [11,12]. In addition, proteins may be (vacuolar protein sorting) mutants secrete the soluble sorted to endosomes via several pathways. Perhaps, the vacuolar hydrolase carboxypeptidase Y (CPY), vam best-characterised route from the late Golgi to the late mutants have abnormal vacuole morphology, pep mutants endosome or multivesicular body (MVB, also referred to as are defective for vacuolar protease activity, vac mutants the prevacuolar compartment) is named after its most studied cargo CPY. Another route takes proteins from the late Golgi, by-passing the endosomal network, to the * Corresponding author. vacuole, and is again named after its most studied cargo E-mail address: [email protected] (T.H. Stevens). protein, alkaline phosphatase [ALP, see Refs. [13–15]]. A

Table 1 Table 1 (continued) VPS genes Alias ORF Refs. Alias ORF Refs. designation designation Class E VPS genes Class A VPS genes VPS25 – YJR102C [3–5] 1 VPS8 FUN15 YAL002W [1–3,5,99] VPS27 GRD11, DID7, YNR006W [3,5,10,153] VPS10 PEP1 YBL017C [2,3,5,7] SSV17 VPS13 SOI1 YLL040C [2,3,5,31,178] VPS28 – YPL065W [3–5] VPS29 PEP11 YHR012W [2,3,5,7] VPS31 BRO1, LPF2, YPL084W [3,5,216–218] VPS30 APG6, ATG6 YPL120W [3,5,191,192] ASI6, NPI3 VPS35 GRD9 YJL154C [2,3,5,10] VPS32 SNF7, DID1 YLR025W [1,3,5,153,214] VPS38 – YLR360W [4,5,178] VPS36 VAC3, GRD12 YLR417W [4,5,9,10,178] VPS55 – YJR044C [8,188] VPS37 SRN2, SRN10 YLR119W [4,5,219,220] VPS63* – YLR261C [8] VPS44 NHX1, NHA2 YDR456W [5,127,158,221] VPS70 – YJR126C [8] VPS46 DID2, FTI1, CHM1 YKR035W-A [5,130,153,212] VPS74 API1 YDR372C [8] VPS60 MOS10, CHM5 YDR486C [133,212,222]

Class B VPS genes Class F VPS genes VPS5 PEP10, GRD2 YOR069W [2,3,5,7,10] VPS1 GRD1, LAM1, SPO15 YKR001C [1,3,5,10,59,223] VPS17 PEP21 YOR132W [2,3,5] VPS26 PEP8, GRD6 YJL053W [2,5,7,10] VPS39 VAM6, CVT4 YDL077C [4–6,193] VPS62 – YGR141W [8] VPS41 VAM2, CVT8, YDR080W [5,6,193–195] VPS65 – YLR322W [8] FET2, SVL2 VPS68 – YOL129W [8] VPS43 VAM7 YGL212W [5,6] VPS711 SWC6 YML041C [8,203] VPS51 VPS67, WHI6, API3 YKR020W [184,196,197] VPS genes and their alternative names are shown. Two sets of initial screens VPS52 SAC2 YDR484W [183,198] were performed to identify vps mutants. One set identified vpt mutants VPS53 – YJL029C [183] [2,3], and the other vpl mutants [1,4,5]. Complementation testing and VPS54 LUV1, CGP1, TCS3 YDR027C [183,199–201] renaming generated the VPS nomenclature [see Ref. [5]], and hence the vpt VPS61* – YDR136C [8] and vpl names have been omitted. Other names, as shown in the VPS64 FAR9 YDR200C [8,202] Saccharomyces genome database [190], are included. 1VPS8 and VPS71 VPS66 – YPR139C [8] have been put into two classes. *Denotes open reading frames that may not VPS69* – YPR087W [8] encode proteins (dubious ORFs) and that when deleted are likely to affect VPS711 SWC6 YML041C [8,203] neighbouring genes involved in CPY trafficking [8,190]. VPS72 SWC2 YDR485C [8,203] VPS73 – YGL104C [8] third route takes proteins from the late Golgi to early VPS75 – YNL246W [8] endosomes [16,17]. Class C VPS genes VPS11 PEP5, END1, YMR231W [3–7,18,204] VAM1 2. Vacuolar protein sorting mutants VPS16 VAM9, SVL6 YPL045W [3,5,6,18,194] VPS18 PEP3, VAM8 YLR148W [3,5–7,18] VPS33 PEP14, VAM5, YLR396C [3,5–7,18,205–207] 2.1. vps mutants secrete the soluble hydrolase CPY and are MET27, CLS14, classified based on vacuolar morphology SLP1 CPY is synthesized as a prepro form and is transported Class D VPS genes across the endoplasmic reticulum (ER) membrane. Follow- VPS3 PEP6 YDR495C [1,3,5,7] VPS6 PEP12 YOR036W [1,3,5,7] ing signal peptidase cleavage in the ER to produce a 67 kDa VPS81 FUN15 YAL002W [1–3,5,99] precursor (p1) form, it is transported through the Golgi VPS9 – YML097C [3,5] where it acquires sugar modifications to become a 69 kDa VPS15 VAC4, GRD8 YBR097W [3–5,9,10] (p2) form. In wild-type cells, CPY is transported to the VPS19 PEP7, VAC1 YDR323C [3,5,7,9] vacuole where it is processed to a 61 kDa mature form. VPS21 YPT51 YOR089C [3,5,208] VPS34 PEP15, END12 YLR240W [1,3,5,7,209] However, in vps mutant cells, a portion of the p2 (Golgi) VPS45 STT10 YGL095C [5,210] form of CPY is secreted from the cell. There are currently 61 yeast VPS genes (see Table 1), and the majority of these Class E VPS genes have been identified in screens designed to find mutants that VPS2 REN1, GRD7, YKL002W [1,3,5,153,211,212] secrete CPY [1–5,8]. It should be noted that whereas all vps DID4, CHM2 VPS4 END13, DID6, YPR173C [1,3,5,10,153, mutants secrete CPY, there are many other mutants that GRD13, CSC1 209,213] secrete low levels of this protein. For example, a recent VPS20 CHM6 YMR077C [3–5,212] screen of the yeast genome deletion collection identified VPS22 SNF8 YPL002C [3–5,214] 146 genes that when deleted caused CPY secretion [8]. VPS23 STP22 YCL008C [3–5,215] These included the previously identified VPS genes, many VPS24 DID3 YKL041W [3,5,153] genes involved in other cellular processes, and some 440 K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454

Table 2 Classification of vps mutants Class Vacuolar morphology A Wild-type: 3–10 spherical vacuoles that cluster in one area of the cytoplasm. In dividing cells, the vacuole extends from mother to daughter cell along the cell axis. B Fragmented vacuoles: more than 20 small, vacuole-like compartments. C No identifiable vacuoles. D Single, large vacuole that fails to extend into daughter cell buds. E Vacuoles larger than wild-type, with a very large, aberrant late endosome/MVB or prevacuolar compartment (the class E compartment) adjacent to the vacuoles. F One large vacuole surrounded by a number of fragmented vacuolar structures. For further details, see Ref. [5].

Fig. 1. Protein trafficking pathways from the late Golgi to the vacuole in endosome, CPY dissociates from Vps10p and Vps10p is yeast. There are multiple protein transport pathways from the late Golgi. Proteins can be secreted or targeted to the cell surface, or transported to returned to the late Golgi via a retrograde transport pathway early endosomes (EE). In addition, the CPY pathway takes proteins via the that requires the retromer complex and probably Vps1p late endosome/multivesicular body (MVB) to the vacuole. The CPY [23,24]. CPY is transported to the vacuole where it is pathway and endocytic pathways converge at or before the late endosome/ proteolytically activated to its mature form. The receptor- MVB. Transmembrane proteins destined for the vacuole lumen such as mediated trafficking of Vps10p is shown in Fig. 2. Ste2p (which is degraded in the vacuole) or CPS (a vacuolar hydrolase) are sorted into the inner membranes of the MVB. Proteins destined for the The intracellular trafficking of Vps10p is mediated by its limiting membrane of the vacuole such as subunit a of the V-ATPase cytosolic domain [20,25]. Within this domain, two aro- (Vph1p) are not sorted into these lumenal vesicles. The ALP pathway sorts matic-based signals, YSSL and FYVF, enable cycling of the proteins such as ALP and Vam3p to the vacuole from the late Golgi, protein between the late Golgi and the endosome. Whereas bypassing the endosomal network. the cytosolic domain controls trafficking, it is the lumenal portion of Vps10p that binds ligands. CPY, PrA, and unknown open reading frames. However, some of the open aminopeptidase Y bind the same site of a region named reading frames identified in this screen are unlikely to ‘‘domain 2’’ between a cysteine-rich domain and the encode proteins, and the phenotypes are likely due to the transmembrane domain (and encompassing a second cys- disruption of adjacent or overlapping genes involved in teine-rich domain) [21]. Interestingly, fusions of CPY- protein trafficking [see Table 1 and Ref. [8]]. invertase and PrA-invertase bind to a different site in The vps mutants have been divided into six classes (A– ‘‘domain 2’’. F) based on several criteria, including the morphology of the In addition to Vps10p, yeast cells have a Vps ten mutant vacuoles [5,18]. This classification has proved very homologous protein, Vth2p [20]. Vth2p is encoded by two useful since mutants that fall into the same class are often almost identical genes, VTH1 and VTH2 (they differ in 1 blocked in the same step of protein trafficking, and in many base pair). Overexpression of VTH2 partially suppresses cases, the proteins encoded by genes in the same class have secretion of CPY and PrA in vps10D cells and thus Vth2p is been found to form complexes (see below). The morpho- capable of sorting both CPY and PrA. However, logical characteristics of the vps classes are described in vps10Dvth1Dvth2D cells are still able to sort a large portion Table 2. of PrA suggesting that the Vps10p-independent sorting of

2.2. Vps10p is the CPY receptor

The receptor that controls the trafficking of CPY out of the late Golgi and to the late endosome was identified in 1994 as the product of the VPS10/PEP1 gene [19]. Vps10p is made up of 1577 amino acids, and is a type I membrane protein, localised predominantly to the late Golgi at steady state. Cells lacking Vps10p secrete more than 90% of newly synthesized CPY and 50% and 15%, respectively, of the vacuolar hydrolases proteinase A (PrA, Pep4p), and amino- Fig. 2. Receptor-mediated trafficking of CPY. CPY binds its receptor peptidase Y [19–21]. The CPY/Vps10p complex is trans- Vps10p and the CPY/Vps10p complex is sorted from the late Golgi to the late endosome (LE). CPY dissociates from Vps10p at the late endosome, ported from the late Golgi to the late endosome in a and is transported to the vacuole, where it is cleaved by proteases to vesicular trafficking step that involves both clathrin and the generate the mature form (mCPY) from the Golgi-modified p2 form. dynamin-related GTPase Vps1p [22]. On arrival at the Vps10p is recycled back to the Golgi for further rounds of CPY sorting. K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454 441

PrA is carried out by another receptor. One candidate for pathways. This partitioning may be controlled by Vps13p/ such a receptor is a protein with weak homology to the Soi1p (a negative regulator of TLS2) [31,33]. Vps13p is mammalian mannose-6-phosphate receptors, Mrl1p [26]. also thought to act as a positive regulator of Kex2p retrieval Deletion of both MRL1 and VPS10 increased missorting of from the late endosome to the late Golgi [31]. PrA to 80%, and Mrl1p cycles through a late endosomal As shown in Fig. 1, the ALP pathway to the yeast compartment from the late Golgi, as would be expected of a vacuole by-passes endosomes. ALP and Vam3p (another vacuolar hydrolase sorting receptor. However, there is cargo of this pathway) both contain acidic dileucine sorting currently no evidence for direct binding of vacuolar hydro- signals in their cytoplasmic domains that are required for lases to Mrl1p, and it remains possible that Mrl1p plays a packaging these proteins into transport vesicles [35–37]. more indirect role in their sorting. The dileucine signals associate with the AP3 adaptor complex for sorting at the late Golgi (see below).

3. Exit from the late Golgi to endosomes or the vacuole 3.2. Clathrin and adaptor proteins for vesicular transport at the late Golgi 3.1. Cargo sorting at the late Golgi Vesicular transport from the late Golgi to endosomes As shown in Fig. 1, there are several different destina- requires both clathrin and clathrin-adaptor proteins. The tions for proteins exiting the late Golgi. Proteins must clathrin coat is required as a scaffold for vesicle budding, therefore be sorted at the late Golgi for transport to the and the adaptor proteins bind both clathrin and cargo to link correct location. As described above, the CPY receptor vesicle formation with protein sorting [for review, see [38]]. Vps10p is transported to the endosome and sorted back to Transport along the CPY pathway, from the late Golgi to the the late Golgi by a retrieval process. Two other well-studied late endosome requires clathrin and the adaptors Gga1p and cargo proteins are the mating pheromone a-factor proces- Gga2p [reviewed in [39]]. Deletion of both GGA1 and sing enzymes Kex2p and dipeptidyl aminopeptidase A GGA2 results in a VpsÀ phenotype, although deletion of (DPAP A, also known as Ste13p). Much of the information GGA1 or GGA2 alone does not [40,41]. This explains why on the trafficking of DPAP A comes from studies of a the GGAs were not isolated from the vps mutant screens. chimeric protein consisting of the cytosolic domain of Recent data suggest that the Gga proteins bind ubiquitin to DPAP A fused to the transmembrane and lumenal domains facilitate sorting of ubiquitinated cargo such as the general of ALP (A-ALP) [27]. Like Vps10p, Kex2p and DPAP A amino acid permease Gap1p from the late Golgi to the late have aromatic-based amino acid motifs that associate with endosome/MVB [42,43]. Studies in both yeast and mam- the retromer complex for retrieval to the late Golgi (see malian cells also suggest that Gga proteins may have a role below) [27–29]. However, unlike Vps10p, Kex2p and in the endocytic pathway, perhaps in the sorting of DPAP A have signals for retention in the late Golgi. In A- ubiquitinated proteins at the MVB [42,44–46]. ALP, deletion of the region between amino acids 2 and 11 Unlike transport along the CPY pathway, vesicles trans- results in accelerated delivery to the late endosome [30]. porting ALP to the vacuole from the late Golgi do not need Kex2p has a similar region required for Golgi retention, clathrin. ALP and Vam3p (another cargo protein of this known as TLS2 (trans-Golgi network localisation sequence pathway) require the AP3 adaptor complex for trafficking to 2) [31]. A-ALP and Kex2p are phosphorylated in vivo, and the vacuole. The yeast AP3 adaptor complex consists of 4 phosphorylation of S13 in A-ALP is required for its proteins: Apl6p (a homologue of mammalian h adaptin), trafficking to the late endosome [32]. These data imply that Apl5p (a y adaptin homologue), Apm3p (a A subunit phosphorylation may influence the association of late Golgi homologue), and Aps3p (a j subunit homologue) proteins with the transport machinery. [15,47,48]. Vps41p, a class B Vps protein, associates with Recent data suggest that both Kex2p and A-ALP travel to yeast AP3 and participates in the formation of AP3 vesicles the late endosome via the early endosome and that sorting to from the late Golgi. Vps41p also associates with the Vps the early endosome from the late Golgi requires clathrin and class C complex of proteins that is thought to act at later the synaptojanin-like protein Inp53p [16,33,34]. However, steps in the transport pathway (see below) [49,50]. Thus, mutations in the GTPase Vps1p cause A-ALP and Kex2p to Vps41p on the transport vesicle may provide a link from be targeted to the vacuole via the cell surface suggesting that AP3-dependent cargo selection to eventual docking and there is a Vps1p-dependent late Golgi-endosome step in fusion with the target membrane [51]. their trafficking. Vps1p is thought to be involved in late Another adaptor complex functioning in late Golgi/ Golgi to late endosome trafficking and thus A-ALP and endosome trafficking is the AP1 complex. Like AP3, this Kex2p are likely to travel both directly to the late endosome complex consists of 4 subunits: Apl2p (a h adaptin and indirectly to the late endosome via the early endosome. homologue), Apl4p (a g adaptin homologue), Apm1p (a A For Kex2p, there is some indication that the TLS2 signal is chain homologue), and Aps1p (a j chain homologue) involved in partitioning of the protein between the late [47,48,52]. However, unlike AP3, AP1 interacts both Golgi to early endosome and late Golgi to late endosome physically and genetically with clathrin [48,52–54]. Dele- 442 K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454 tion or mutation of individual AP1 subunits, or deletion of may play a role in regulating peroxisome number, size, and the whole complex in yeast results in no measurable protein distribution [65]. trafficking phenotype. However, in cells with a temperature- sensitive clathrin heavy chain mutation (chc1-ts), mutation or deletion of AP1 subunits results in growth defects at high 4. Membrane fusion at the late endosome/MVB and at temperatures and a-factor maturation defects at all temper- the vacuole atures [52–55]. Deletion of AP1 subunits also exacerbates the growth and trafficking phenotypes of gga mutants, As shown in Fig. 1, the ALP pathway by-passes the late suggesting that AP1 and Gga proteins may control parallel endosome or MVB. This section therefore focuses on the pathways out of the late Golgi [56,57]. Despite this, the fusion of CPY pathway vesicles derived from the late Golgi, exact site of AP1 function remains unclear with good early endosome-derived membranes, and retrograde vesicles evidence for it acting at the late Golgi (to direct transport to from the vacuole with the late endosome/MVB outer the early endosome) and/or at the early endosome (to direct membrane. In addition, we will discuss the role of Vps transport back to the late Golgi) [17,34]. proteins in the fusion of late endosome-derived membranes and ALP pathway vesicles with the vacuolar membrane. 3.3. Vps1p is a dynamin-related GTPase The class D vps mutants have a single, large vacuole, and many accumulate 40–60 nm vesicles as seen by electron Vps1p is an 80 kDa protein with homology to the microscopy [66,67], suggesting that the gene products mammalian GTPase dynamin, although it lacks the proline- defective in these mutants are required for vesicle fusion rich domain and pleckstrin-homology domains of classical events. Membrane fusion reactions require SNARE pro- dynamins [for review, see [58]]. However, it is likely to teins, with each fusion reaction involving an R-SNARE on function in a similar way to dynamin in pinching off one membrane and QA-, QB-, and QC-SNAREs on the other vesicles after budding (vesicle scission). Vps1p is required [68,69]. As well as SNARE proteins, SM (Sec1p/Munc18) for the exit of proteins along both the CPY and ALP proteins, small GTPases of the Rab family, and membrane pathways in yeast, and cells lacking Vps1p are able to tethering factors are necessary for membrane fusion. The transport proteins to the vacuole via the plasma membrane class D Vps proteins include the QA-SNARE Pep12p/ [59,60]. Kex2p, A-ALP, and Vps10p are also delivered to Vps6p, the SM protein Vps45p, the Rab GTPase Vps21p, the vacuole via the plasma membrane in vps1D cells the Rab exchange factor Vps9p, and Rab effector Vac1p/ [24,59,60]. This suggests a role for Vps1p not only in Vps19p/Pep7p. Tethering factors are provided by a complex vesicle budding from the late Golgi, but also in the budding of the class C Vps proteins [for review, see [70]]. of vesicles and retrieval of proteins from endosomes. The other implication from these data is that there is no 4.1. The QA-SNARE Pep12p is involved in multiple fusion requirement for Vps1p for endocytosis from the cell surface, events at the late endosome/MVB or for trafficking through the endocytic pathway in yeast. The kinetics of internalisation of the uracil permease Fur4p The QA-SNARE Pep12p is localised to the late endo- are normal in vps1D yeast at 30 -C, again suggesting no some, and is likely to be involved in the fusion of vesicles at absolute requirement for Vps1p in internalisation from the this compartment [71,72]. Pep12p is involved in several cell surface [61]. There are 3 possible explanations for this: fusion events: the fusion of vesicles originating from the late (1) A dynamin-like GTPase is not required for these Golgi, the early endosome, and the vacuole with the late trafficking steps in yeast. (2) Vps1p is required under endosome/MVB [73–75]. The ability of Pep12p to partic- normal circumstances, but other (perhaps clathrin-independ- ipate in these different fusion events is likely due to its ent) pathways compensate in vps1D cells. (3) Other inclusion in several different SNARE complexes. Pep12p (a dynamin superfamily proteins may play roles in yeast QA-SNARE) appears able to form complexes with Vti1p (a endocytic vesicle scission. In fact, there are 3 other QB-SNARE), Syn8p or Tlg1p (QC-SNAREs), and Snc1p, dynamin-related GTPases in yeast: Dnm1p, Mgm1p, and Snc2p, or Ykt6p (R-SNAREs) [76–78].Pep12palso Fzo1p. These 3 proteins coordinate mitochondrial fission interacts with the yeast N-ethylmaleimide-sensitive factor and fusion, though Dnm1p may also play a role in the (NSF) homologue Sec18p, an ATPase required for the endocytic pathway at a post-internalisation step [see [58] disassembly of SNARE complexes to allow recycling for and references therein, and [62]]. Thus, the role for further rounds of membrane fusion [79,80]. dynamin-like proteins in endocytosis in yeast remains unclear. 4.2. Vps45p and Vps33p are SM proteins required for fusion Very recently, a novel role for Vps1p has been proposed with the late endosome/MVB in vacuole–vacuole homotypic fusion [[63], reviewed in [64]]. As well as roles in protein trafficking, Vps1p is Vps45p is an SM family protein, as is Vps33p (part of involved in the organisation of the actin cytoskeleton and the large class C Vps protein complex, see below). SM binds the actin regulatory protein Sla1p [61]. Finally, Vps1p proteins bind to QA-SNAREs (also called syntaxins), and K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454 443 although essential for membrane fusion reactions, their Pep12p and Vps33p [83,90]. Vac1p also physically and precise role remains a matter of debate [for reviews, see genetically interacts with Pep12p and the GTP-bound form [81,82]]. The QA-SNARE Pep12p interacts both physically of Vps21p, genetically interacts with Vps33p, and binds and genetically with both Vps33p and Vps45p [80,83]. phosphatidylinositol 3-phosphate (PI3P) via its FYVE However, overexpression of VPS45 does not suppress the domain [95–97]. The class C Vps complex has also been trafficking phenotypes of vps33D cells and vice versa, shown to interact with Vps8p, a protein required for protein suggesting that Vps45p and Vps33p have unique roles in transport between the late Golgi and endosomes and protein trafficking [83]. functionally associated with Vps21p [83,98,99]. Vps33p is the SM protein required for the fusion of As discussed above, the class C proteins act at multiple membranes derived from early endosomes with the late steps in vacuolar protein trafficking and the class C Vps- endosome, however, it appears that both Vps33p and phenotype is a result of inhibiting these steps. In contrast, Vps45p are required for the fusion of late Golgi-derived the class D Vps proteins Pep12p, Vps9p, Vac1p, Vps21p, vesicles with the late endosome [83,84]. As well as Pep12p, and Vps45p (see above) and Vps15p and Vps34p (see Vps45p also interacts with another QA-SNARE Tlg2p, and Section 6.2) control aspects of protein trafficking at the level in fact, Tlg2p appears to provide the major membrane of the endosome. Thus, the remaining class D Vps protein, binding site for Vps45p [85,86]. Vps45p dissociates from Vps3p, may act in protein sorting into or out of the Tlg2p before membrane fusion and reassociates with the endosome. Like pep12, vps21 and vps45, vps3 is syntheti- cis-SNARE complex formed after fusion, and is thought to cally lethal with apm3 (which encodes the medium chain of act as a molecular switch for the SNARE complex of Tlg2p, AP3) at high temperatures, suggesting that they act in Tlg1p, Vti1p, and Snc2p [85,87]. Like Vps45p, Vps33p is parallel pathways [15]. However, Vps3p remains one of the associated with more than one QA-SNARE and functions least characterised of the Vps proteins and further work will with Pep12p at the late endosome and Vam3p at the vacuole be needed to determine its precise role in protein trafficking. [83,88].

4.3. The class C Vps proteins make up tethering complexes 5. Protein sorting at the late endosome/MVB for membrane fusion The late endosome/MVB is characterised by the presence Tethering factors include several multisubunit complexes of inner membranes or vesicles (see Fig. 1). On arrival at the and are thought to link the membranes prior to fusion. One MVB, transmembrane proteins are again sorted from one such tethering complex is made up of the class C Vps another: into the inner membranes for transport to the proteins, Vps11p/Pep5p, Vps18p/Pep3p, Vps16p, and vacuole lumen, into retrograde transport vesicles, or they Vps33p. This complex is known as the class C Vps remain on the outer MVB membrane for transport to the complex. The class C Vps complex acts at two distinct limiting vacuolar membrane. protein trafficking steps: fusion of late Golgi vesicles with the late endosome; and fusion at the vacuole [89–91]. 5.1. Ubiquitinated proteins are sorted to the inner The last step of both the CPY and the ALP pathways (i.e. membranes of MVBs fusion with the vacuole) is controlled by a SNARE complex of Vam3p (QA), Vti1p (QB), Vps43p/Vam7p (QC), and One signal for the sorting of proteins into the inner Ykt6p (R), as well as the class C complex and the Rab membranes of multivesicular bodies is the addition of GTPase Ypt7p [76,92]. The class C complex at the vacuole ubiquitin [for review, see [100]]. In most cases, this appears (also called the homotypic fusion and protein sorting to be monoubiquitin, though there is evidence that (HOPS) complex) interacts physically and genetically with polyubiquitination of the general amino acid permease Vam3p and Ypt7p [49,88,89,93]. Two of the class B Vps Gap1p influences its endosomal trafficking [101,102]. There proteins also associate with the class C complex at the is also evidence for a ubiquitin-independent pathway for vacuole: Vps39p/Vam6p and Vps41p. Vps39p stimulates sorting into lumenal MVB vesicles, as characterised by the nucleotide exchange on Ypt7p and thus the class C complex protein Sna3p [103]. Proteins, such as the G-protein- is thought to act as a Ypt7p effector that tethers transport coupled pheromone receptors, Ste2p and Ste3p, are mono- vesicles to the vacuole [49,94]. ubiquitinated at the cell surface, and the ubiquitin acts as a By analogy with the function of the vacuolar class C Vps signal for internalisation as well as sorting at the MVB complex, it is likely that the class C complex at the late [103–109]. Other cargo proteins travel from the late Golgi endosome associates with SNAREs and a Rab GTPase. to the MVB and are ubiquitinated en route, either at the late Thus, Vps21p, Vps9p, Vac1p, and Pep12p may act with the Golgi or the MVB [42,110,111]. Interestingly, many class C Vps complex in vesicle fusion at the late endosome. components of the trafficking machinery for late Golgi to In support of this idea, there is a genetic interaction between endosome or vacuole protein transport have ubiquitin- a mutant allele of vps21 and vps11, vps16,orvps18 binding domains. The Gga proteins bind ubiquitin and temperature-sensitive mutants, and an interaction between mediate the trafficking of some ubiquitinated cargo such as 444 K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454

Gap1p from the late Golgi to the MVB. In addition, Gga (via charged residues in their transmembrane domains) proteins are likely to be involved in MVB protein sorting and [121]. a yeast two-hybrid interaction has been shown between Tul1p has also been implicated in the ubiquitination of mammalian Gga3 and TSG101 (the mammalian homologue proteins at the late Golgi. Tul1p is a transmembrane of Vps23p, a component of ESCRT I, see below) [42,46]. ubiquitin ligase, thought to be involved in the ubiquitination The Vps21p exchange factor Vps9p also binds ubiquitin (via of proteins with polar residues in their transmembrane a CUE domain), and may also be involved in the transport of domains such as CPS, Phm5p, and misfolded proteins [122]. ubiquitinated cargo proteins to the MVB [112–114]. However, while one study shows that tul1D cells have Rsp5p is the only yeast member of the Nedd4 family of severe defects in the ubiquitination and MVB sorting of ubiquitin ligases, and is required for the ubiquitination of CPS and Phm5p [122], others report no effects on MVB proteins at the cell surface, as well as biosynthetic cargo sorting of these cargos in tul1D cells [110,111]. These destined for the vacuole along the CPY pathway findings may be attributable to strain differences, and of [101,102,110,111,115]. Rsp5p is localised to many sites in particular note is the finding of Katzmann et al. that the the cell, including the plasma membrane, Golgi, and EUROSCARF/Invitrogen deletion collection wild-type endosomes [110,116]. Interestingly, different mutations in strain BY4742 has a partial defect in GFP–CPS sorting Rsp5p affect different cargos. For example, deletion of the [110]. C2 domain affects biosynthetic cargo such as carboxypeptidase S (CPS), but does not affect ubiquitination and 5.2. Class E Vps proteins are involved in protein sorting at internalisation of Ste2p at the plasma membrane the MVB [110,111,117]. The C2 domain binds phosphoinositides and is likely to be a determinant in the recruitment of Over the last few years, it has become apparent that the Rsp5p to Golgi and/or endosomal membranes [111]. class E Vps proteins are involved in protein sorting into the Different point mutations in the HECT domain (the domain lumenal vesicles of the MVB. The class E vps mutants are required for ubiquitin transfer or E3 activity) also affect characterised by the presence of a large, aberrant MVB or different trafficking steps. The G555D or G753V mutations late endosome (the ‘‘class E compartment’’) where proteins affect CPS sorting, while L733S affects Ste2p internal- en route to the vacuole via the CPY pathway or the endocytic isation [110,117,118]. The differing effects of these muta- pathway accumulate [see Fig. 3 and [5,123,124]]. The class tions may be due to the role of the HECT domain in E compartment appears as a multilamellar structure of substrate recognition [119,120]. In addition to substrate curved membrane stacks by electron microscopy, and the binding, Rsp5p binds several accessory proteins that may be vacuoles of class E mutants appear devoid of lumenal required for the ubiquitination of specific cargos. For vesicles [124–126]. Late Golgi proteins such as Vps10p also example, Bul1p and Bul2p bind Rsp5p and are required accumulate in the class E compartment, since recycling to the for the ubiquitination-dependent sorting of Gap1p and the late Golgi is inhibited in class E vps mutants [25,123,124]. tryptophan permease Tat2p, but not for the sorting of CPS, Together, these data suggest that the class E mutants are Ste2p, or Fur4p. More recently, a three-transmembrane unable to form and sort proteins into lumenal MVB vesicles, domain protein, Bsd2p, has been proposed to link Rsp5p and inhibited in the exit of proteins from the MVB (via its WW domains) to the substrates CPS, the poly- compartment. However, the inhibition of recycling of Golgi phosphatase Phm5p, and the manganese transporter Smf1p proteins in the class E vps mutants may not necessarily imply

Fig. 3. Protein trafficking in class E vps mutant cells. (a) In wild-type cells, Ste2p is internalised from the plasma membrane, transported through the early endosome and incorporated into the inner membranes of the MVB. On fusion of the late endosome/MVB with the vacuole, Ste2p is degraded by vacuolar proteases. The pro form of CPS (pCPS) is transported from the late Golgi to the MVB, into the lumenal vesicles and subsequently to the lumen of the vacuole where it is matured (mCPS). Vps10p cycles between the late Golgi and the late endosome/MVB. (b) In class E vps mutants cells, Ste2p, pCPS, and Vps10p accumulate in the large, aberrant endosomal structure, the class E compartment. Transport from the class E compartment to the vacuole is inhibited, asis retrograde transport of Vps10p back to the late Golgi. The Vps10p accumulated in the class E compartment undergoes aberrant proteolytic cleavage to a smaller form (Vps10p*). K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454 445 that the class E proteins play a direct role in this transport dylcholine, which accumulates specifically in the lumenal step. Inhibition of retrograde transport could be a conse- membranes). Cells expressing UIM-mutated ESCRT 0 are quence of the aberrant multilamellar membranes of the class also able to recycle Vps10p to the late Golgi and sort CPY, E compartment. The defect in Vps10p recycling is probably but unable to correctly sort Ste3p, Ste2p, and CPS to the the major cause of the CPY secretion phenotype, since vacuole [126,135]. Thus, UIM mutants of ESCRT 0 Vps10p is trapped in the class E compartment and is provide the first indication that MVB formation (i.e., the unavailable for further rounds of CPY sorting. This is formation of lumenal vesicles) and recycling out of the supported by evidence that overexpression of Vps10p (and MVB can be separated from the sorting of ubiquitinated thus increasing the amount of Vps10p at the late Golgi cargo [126]. independent of recycling) suppresses the CPY secretion of As well as interacting with ubiquitinated cargo, ESCRT 0 vps27D, vps4, vps23,orvps28 cells [123,125].The binds the lipid PI3P via the FYVE domain of Vps27p and accumulation of Vps10p in the class E compartment in the ESCRT I protein Vps23p [via PTAP-like motifs in the mutant cells also results in more rapid processing to a smaller C-terminal region of Vps27p, [136,137]]. The activity of the form, Vps10*, than is seen in wild-type cells [see Fig. 3 and phosphatidyl inositol 3-kinase Vps34p is required for the [25,123,127]]. synthesis of PI3P in yeast, and in vps34D cells, Vps27p is 16 class E VPS genes are shown in Table 1. In addition to not recruited to membranes [137]. Thus, ESCRT 0 is these, there are two further genes, HSE1 and VTA1, that also proposed to associate with the membrane via binding fall into this class, but were not given VPS designations ubiquitinated cargo and PI3P and recruit ESCRT I. More [126,128,129]. Two of the class E vps mutants, vps44 and recently, an interaction has been shown between Vps27p vps46, were originally classified as class A mutants but and the ENTH domain proteins, Ent3p and Ent5p [138]. further analysis and the availability of a wider range of These epsin-family proteins also bind phosphatidyl inositol- morphological markers has led to their reclassification 3, 5-bisphosphate, a lipid required for the sorting of a subset [5,127,130]. of proteins at the MVB and the product of the Fab1p phosphatidyl inositol 3-phosphate 5-kinase enzyme [138– 5.3. The soluble class E Vps proteins form complexes with 142]. Ent3p and Ent5p have overlapping function at the one another MVB and are required for the correct sorting of proteins to the lumenal membranes [138]. It has been proposed that Of the 18 proteins encoded by class E VPS genes, 17 are Ent3p and Ent5p may deform the MVB membrane during soluble or peripherally membrane-associated proteins. Only vesicle formation, in a similar way to the proposed function one, Vps44p/Nhx1p, is a transmembrane protein (see of epsin 1 at the plasma membrane of mammalian cells below). Biochemical and genetic analyses of the interactions [138,143]. However, the topology of membrane curvature is of the soluble class E Vps proteins have identified four reversed at the MVB (the vesicles bud away from the endosomal sorting complexes required for transport (ESCRT cytosol and into the lumen), and further experiments will be complexes): Vps27p and Hse1p [ESCRT 0, named after the required to determine the precise function of Ent3p/Ent5p at equivalent complex in mammalian cells, [126,131]], the MVB. Vps23p/Stp22p, Vps28p, and Vps37p/Srn2p [ESCRT I, As well as effecting the recruitment of ESCRT I to MVB [105]], Vps22p/Snf8p, Vps25p, and Vps36p [ESCRT II, membranes, ESCRT 0 may interact with the ubiquitin ligase [132]], and Vps2p/Did4p, Vps24p, Vps20p, and Vps32p/ Rsp5p. Yeast two-hybrid studies have shown an interaction Snf7p [ESCRT III, [133]]. The remaining soluble class E between Hse1p and Rsp5p, though this has not been Vps proteins (Vps4p, Vps31p/Bro1p, Vps46p/Did2p, confirmed yet by other techniques [130]. However, Rsp5p Vps60p, and Vta1p) are all associated with ESCRT III has been localised to endosomes and is required for the [130,134]. ubiquitination of cargo molecules, such as CPS (see above). ESCRT 0 may therefore recruit Rsp5p to the MVB 5.4. The ESCRT 0 complex binds ENTH domain proteins, membrane, allowing ubiquitination of cargo. Ubiquitinated ubiquitinated cargo proteins, and ESCRT I cargo would then be positioned to immediately bind ESCRT 0 and trigger ESCRT-mediated MVB sorting. The ESCRT 0 complex of Vps27p and Hse1p is the sorting receptor for ubiquitinated cargo proteins at the MVB 5.5. ESCRT I binds ubiquitinated cargo, as well as ESCRTs (see Fig. 4). Both Vps27p and Hse1p contain ubiquitin II and III interacting motifs (UIMs), and these motifs bind ubiquitin in vitro and are essential for the function of ESCRT 0 in MVB As described in the previous section, ESCRT 0 binds sorting [126,135]. Interestingly, while vps27D or hse1D ESCRT I via Vps27p–Vps23p interactions [130,136,137]. cells have a VpsÀ class E phenotype, cells expressing In addition, there is a yeast two-hybrid interaction between Vps27p and Hse1p with UIM mutations are able to make Hse1p and Vps23p [130]. Vps23p also binds ubiquitinated lumenal vesicles (as assayed by electron microscopy and the cargo, but unlike ESCRT 0, its interaction with ubiquitin is dye 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labelled phosphati- via a ubiquitin E2 variant (UEV) domain [105]. In addition 446 K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454

Fig. 4. Protein sorting at the MVB. (a) Phosphatidyl inositol 3-phosphate (PI3P) is generated from phosphatidyl inositol by the Vps34p/Vps15p complex. ESCRT 0 is recruited to the MVB membrane via PI3P binding. Cargo proteins not previously ubiquitinated may be ubiquitinated by Rsp5p recruited by ESCRT 0. (b) ESCRT 0 binds ubiquitinated cargo and recruits ESCRT I, which binds both ESCRT 0 and ubiquitinated cargo. (c) ESCRTs II and III are recruited. ESCRT II binds ubiquitinated cargo as well as ESCRT I, while ESCRT III interacts with ESCRTs I and II. ESCRT III also interacts with lipids via the myristoylation of Vps20p, and the possible binding of Vps24p to PI3,5P2 (the product of the Fab1p lipid kinase). (d) ESCRT III-associated proteins Vps31p, Vps46p, Vta1p (V), and Vps60p bind and the deubiquitinating enzyme Doa4p is recruited by Vps31p. (e) Cargo is deubiquitinated by Doa4p, and the ATPase Vps4p binds ESCRT III. ATP is hydrolysed on Vps4p. (f) The ESCRT complexes and ESCRT III associated proteins dissociate from the membrane and the cargo is sorted into the inward budding vesicle. to cargo-binding, ESCRT I functions to recruit ESCRTs II III and is proposed to increase membrane-binding of and III, and multiple interactions between these complexes ESCRT III via a direct interaction with Vps20p have been identified [see Fig. 4,and[130,132,133], [130,132,145]. Ubiquitinated cargo also interacts with reviewed in [144]]. ESCRT II, via an NZF ubiquitin-binding domain in Vps36p [145,147]. This domain is not only required for binding 5.6. ESCRTII is a ‘‘Y’’-shaped protein complex ubiquitin in vitro, but is also essential for ESCRT II function in vivo, since expression of a vps36 molecule with point The crystal structure of yeast ESCRT II core has recently mutations in this NZF domain (T187G and F188S) fails to been solved by two groups [145,146]. This core of ESCRTII correct the GFP-CPS sorting defect of vps36D cells [147]. contains 2 Vps25p molecules, one Vps22p, and the C- Thus, ubiquitinated cargo is proposed to bind ESCRT 0, terminal domain of Vps36p, and the structure forms a ‘‘Y’’- ESCRT I, and then ESCRT II during protein sorting at the shape. ESCRT II interacts with both ESCRT I and ESCRT MVB [see Fig. 4, and [147]]. K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454 447

5.7. ESCRT III recruits accessory proteins and the would in turn affect ESCRT or ESCRT-associated protein AAA-ATPase Vps4p recruitment to the membrane, or ESCRT inter- or intra- complex interactions. In support of this model, it has ESCRT III is made up of two subcomplexes: Vps2p/ recently been demonstrated that Nhx1p controls the Vps24p and Vps20p/Vps32p [133]. Myristoylation of the lumenal pH of endosomes and that addition of a weak N-terminal glycine of Vps20p is important for the base to nhx1D cells suppresses some of their protein- membrane association and formation of ESCRT III, and trafficking phenotypes [159]. Vps20p is also the subunit proposed to interact with Recently, a two-hybrid screen has identified Gyp6p, a ESCRTs I and II [130,132,133,145]. The mammalian GTPase activating protein (GAP) for the Rab/Ypt GTPases homologue of Vps24p is a phosphatidyl inositol-3, 5- as a binding partner for the C-terminal hydrophilic portion bisphosphate (PI3,5P2) binding protein, and it is interesting of Nhx1p [160]. This is a potentially interesting finding, to speculate that yeast Vps24p may also bind this lipid [see since Ypt6p (the preferred substrate of Gyp6p) is involved Fig. 4, and [148]]. Thus, ESCRTIII may be recruited to the in endosome to Golgi transport as well as retrograde MVB membrane via multiple mechanisms including bind- transport through the Golgi and possibly Golgi to ER ing to ESCRTs I and II, lipid modification, and lipid trafficking [161,162]. However, topological studies of both interactions. ESCRT III also interacts directly or indirectly Nhx1p and the homologous Arabidopsis thaliana AtNhx1 with Vps4p, Vps46p, Vps60p, Vta1p, and Bro1p [the show that the C-terminal domain is in the endosomal ESCRT III-associated proteins, see Fig. 4, and [128– lumen [163,164]. Gyp6p is cytosolic, and thus the 130,149,150,151]]. Bro1p has recently been shown to physiological relevance of the interaction with Nhx1p recruit the deubiquitinating enzyme Doa4p to the ESCRT remains unclear. III complex, presumably via the interaction of Bro1p with Vps32p [130,134,152]. Doa4p activity removes ubiquitin from the cargo molecule, a step that is not essential for the 6. Retrograde transport inclusion of cargo into the budding lumenal vesicle, but is essential for the maintenance of normal cellular ubiquitin The dynamic systems of protein and membrane transport levels [153–155]. in the eukaryotic cell require not only mechanisms to send In addition to recruiting Doa4p via Bro1p, ESCRT III also components to the correct destinations, but also efficient recruits the AAA-ATPase Vps4p. Cells lacking Vps4p mechanisms to retrieve them. For example, proteins such as (vps4D), or cells expressing Vps4p with point mutations in SNAREs must be recycled following membrane fusion in the ATPase domain (dominant negative mutants unable to order to maintain their correct cellular localisations and bind or hydrolyse ATP) have increased amounts of the preserve the specificity of the fusion reactions. ESCRT complexes on the MVB membrane [105,125,132, 133,156]. Thus, Vps4p activity is required for the mem- 6.1. Proteins can travel from the vacuole to the late Golgi brane-dissociation of ESCRT complexes for subsequent via the late endosome/MVB recycling from the cytosol in further rounds of MVB protein sorting (see Fig. 4). Studies in mammalian cells have shown that late endosomes and pre-existing lysosomes can exchange 5.8. Nhx1p is a sodium/proton exchanger of the NHE family content by both transient ‘‘kissing’’ events and direct fusion reactions [[165], for reviews, see [166,167]]. Thus, As described above, 15 of the class E Vps proteins, as in yeast, it seems likely that the late endosome or MVB well as Vta1p and Hse1p that also fall into this class, are fuses with the vacuole. This model predicts that proteins soluble proteins that are able to form membrane-associated and membrane must be retrieved following fusion to complexes on the MVB membrane. However, the remain- maintain the distinct composition of the compartments. ing member of this class, Nhx1p, is a transmembrane The first demonstration for such a retrieval pathway in sodium/proton exchanger localised to the MVB [127,157, yeast comes from studies with an ALP chimera [75]. 158]. Nhx1p is the only yeast member of the NHE family of Retrieval signal ALP (RS-ALP) has the sequence sodium/proton exchangers, and its precise role in MVB RRESFQFNDI (including the Golgi retrieval signal) from protein-sorting remains unclear. However, point mutants DPAP A in place of residues 11–17 of ALP. RS-ALP is of Nhx1p predicted to be defective in ion transport are found in the late Golgi at steady state, and analysis of its not able to complement the trafficking phenotypes of trafficking route found that it was transported first to the nhx1D cells, suggesting that it is the ion exchange vacuole along the ALP pathway and is then transported activity of Nhx1p that is essential for MVB sorting via the late endosome to the late Golgi. The transport of [127]. Our current model of Nhx1p function in protein RS-ALP out of the vacuole is dependent on Vac7p. In sorting predicts that the lumenal MVB ionic environment addition to the RS-ALP chimera, the QB-SNARE Vti1p uses may influence the conformation of an as yet unidentified this Vac7p-dependent pathway as part of its normal cellular ion sensor protein. This transmembrane ion sensor protein itinerary. 448 K. Bowers, T.H. Stevens / Biochimica et Biophysica Acta 1744 (2005) 438–454

6.2. The retromer complex controls retrograde protein Ypt6p and the QC-SNARE Tlg1p, and may act by activating traffic from the late endosome to the late Golgi SNARE assembly and therefore promoting membrane fusion [182,184,185,187]. This model suggests that the As discussed in Sections 2 and 3, the localisation of GARP complex links vesicle tethering directly to the many late Golgi proteins is maintained by a process of membrane fusion event. retrieval from endosomes. In addition, the function of the CPY sorting receptor Vps10p requires its constant cycling between the late Golgi and the late endosome/MVB. The 7. Conclusions retromer complex, consisting of Vps35p, Vps29p, Vps26p/ Pep8p, Vps5p, and Vps17p is essential for this retrieval Our review has focussed on the yeast Vps proteins and process [for review, see [168]]. Retromer consists of two the protein trafficking pathways they control. Most Vps subcomplexes: the Vps35p–Vps29p–Vps26p subcomplex proteins now have either a predicted or determined function, which has a role in cargo selection and the Vps5p–Vps17p with yeast trafficking pathways becoming increasingly well subcomplex which has a structural role. Vps35p has been understood. However, there remain a few less well- shown to interact with cargo proteins such as A-ALP and characterised Vps proteins. These include Vps55p, a protein Vps10p, and Vps29p and Vps26p are necessary for the with four transmembrane domains that localises to the late interaction of the two subcomplexes with one another endosome [188]; Vps3p, a class D Vps protein [15,189]; [29,169–171]. Vps5p and Vps17p are members of the and the newly identified Vps61–75 proteins found in a sorting nexin family of proteins and are able to bind PI3P genome-wide screen for vps mutants [8] (see Table 1). via a domain known as a Phox homology (PX) domain Further work will be needed to establish the function of [172]. Yeast has one phosphatidylinositol 3-kinase (PI3 these proteins. kinase), Vps34p, which forms a complex with the protein Almost all of the Vps proteins have mammalian kinase Vps15p [173–175]. Vps34p regulates retromer homologues, and in most cases, these have been shown to function via Vps30p and Vps38p, two proteins that form a play equivalent roles in yeast and mammals cells. For multimeric complex with Vps34p and Vps15p. The com- example, there are mammalian homologues of the retromer plex of Vps34p, Vps15p, Vps30p, and Vps38p is known as complex, ESCRT complexes and class C/HOPS complex. ‘‘complex II’’ and directs the synthesis of PI3P on endo- However, while studies in yeast have provided the key somal membranes that is required for retromer recruitment components and basic mechanisms of endosomal protein [172,176]. A second PI3 kinase complex has also been transport, the specialised cells of multicellular organisms identified (‘‘complex I’’) consisting of Vps34p, Vps15p, will be inevitably more complicated. Nevertheless, the Vps30p, Apg14p, and this PI3 kinase complex functions in identification and characterisation of the yeast Vps proteins autophagy [176]. Thus, there are two PI3 kinase complexes has provided much of the information regarding the directing PI3P synthesis at different sites in the cell. molecular mechanisms of endosomal protein trafficking in As well as the Vps5p and Vps17p components of eukaryotic cells. The information gained from the yeast retromer, several other sorting nexins have been implicated system is currently being used to great advantage in the in retrieval from endosomes. Mvp1 is required for the study of similar systems in mammalian cells. retrieval of Vps10p and Kex2p from endosomes, while Grd19p is needed for the sorting of A-ALP and Kex2p but not Vps10p [177–179]. The crystal structure of both the free Acknowledgements form of Grd19p and the form complexed to PI3P has recently been solved [180]. Three more sorting nexins, Snx3p, We would like to thank Nia Bryant and Matthew Seaman Snx41p, and Snx42p, are involved in retrieval from the for critical reading of the manuscript. We also acknowledge early endosome to the late Golgi of the R-SNARE Snc1p the support of the British Heart Foundation (K. B. is the [181]. recipient of an Intermediate Fellowship), and the National Institutes of Health (grants GM32448 and GM38006, 6.3. GARP/VFT is a tethering complex awarded to T. H. S.).

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