- Email: [email protected]
Protein transport to the yeast vacuole
Protein transport to the yeast vacuole Bruce F Horazdovsky, Southwestern Medical Genetic
Daryll B DeWald
Center, Dallas and University
and biochemical
have identified
analyses
Included among these components
of yeast vacuolar
proteins
Some of these gene products are homologous
localization
pathway.
Recent
each of these components offered new insights
Current
kinase, a
and a dynamin-like
to proteins required for
at other stages of the secretory and endocytic pathways.
appear to be required
localization
protein
San Diego, USA
are a sorting receptor, a protein
kinase, small CTP-binding
sorting and transport Others
of California,
more than 40 gene products that play a role in this process.
phosphatidylinositol CTPase.
and Scott D Emr
for unique studies
functions
have helped
plays in vacuolar
into the molecular
Opinion
The vacuole of the yeast Saccharomyces cerevisiae is functionally equivalent to the mammalian lysosome: both are acidic compartments involved primarily in the degradation of macromolecules [1,2]. The degradative nature of the lysosome/vacuole results 6om the presence of a variety of hydrolytic enzymes that have been specifically delivered to these organelles. This localization process is complex, involving a large number of cellular components that function in the recognition and vesicular transport of these hydrolases. Newly synthesized vacuolar hydrolases transit through the early stages of the secretory pathway as inactive zymogens together with proteins that are destined to be secreted from the cell (see Salama and Schekman, this issue, pp 536-543; [3]). Soluble vacuolar hydrolases are actively sorted away from the secretory protein pool in a late-Golgi compartment (possibly equivalent to the trans-Golgi network) and delivered to the vacuole via an endosomal intermediate [4-6]. Upon arrival in the vacuole, the hydrolases are then processed to their mature active forms. In an effort to identify the tram-acting cellular machinery responsible for this schemes have localization process, several selection been developed to identify mutants that mislocalize sorting vacuolar proteins (ups, for vacuolar protein defective) [7-lo]. In addition, mutants that exhibit decreased vacuolar protease activity have been identified (pep, for peptidase deficient) [l 11, many of which also show defects in vacuolar protein delivery [lo]. Together, the ups and pep mutants define more than 40 complementation groups [5], reflecting the complex nature of the vacuolar protein localization pathway.
protein
1995,
protein
the role that
localization
mechanisms
in Cell Biology
Introduction
in the vacuolar to define
and have
of protein sorting.
7:544-551
Recent analyses of the gene products affected in several of the mutants have provided new insights into the mechanisms involved in vacuolar protein localization [12*]; these developments are the subject of this review.
Receptor molecules
for vacuolar
protein sorting
The role of receptor molecules in vacuolar protein localization had been postulated for many years [13]. This proposal was based primarily on the observation that the vacuolar protein sorting system can be saturated. When vacuolar carboxypeptidase Y (CPY) or proteinase A (PrA) were overproduced, a large portion of the overexpressed protein was secreted from the cell in its Golgi-modified precursor form [13,14]. Therefore, it appeared that a limiting component in the vacuolar protein sorting machinery (such as a receptor molecule) was being saturated and the overproduced vacuolar proteins were being redirected to the cell surface by a default localization pathway. Interestingly, the amino-terminal vacuolar protein localization signals of CPY and PrA share no obvious sequence similarity [l], indicating that two different receptor molecules (one for CPY and one for PrA) may be involved in vacuolar protein localization. To identify the gene encoding the proposed CPY sorting receptor, Marcusson et al. [15”] carefully examined the ups mutant collection and uncovered a small group of mutants (vps10, ups.29 and vps3-5) that showed a CPY-specific sorting defect; CPY was secreted in these mutants whereas the vast majority of PrA was correctly delivered to the vacuole [15**, 161. The gene affected in
Abbreviations ALP-alkaline
544
phosphatase; CPY-carboxypeptidase Y; DPAPB-dipeptidyl aminopeptidase 6; PI-phosphoinositide; PrA-proteinase A; Ptdlntphosphatidylinositol; SNARE-SNAP receptor.
0 Current Biology Ltd ISSN 0955-0674
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
one of these mutants, I/PSI@ encodes a CPY sorting receptor. VpslOp is a type I transmembrane protein, with a 1393 amino acid amino-terminal lumenal domain and a cytoplasmic 164 amino acid carboxy-terminal domain (Fig. 1). It is also a stable protein that cofractionates with a late-Golgi marker protein, placing this receptor molecule at the site where vacuolar proteins are sorted. Most importantly, cross-linking experiments show that VpslOp interacts specifically with precursor CPY and this interaction is mediated by the vacuolar sorting signal of CPY [15**]. At present, vps mutant selections have not uncovered a candidate PrA receptor molecule; however, it is likely that other vacuolar protein sorting receptors are present in this pathway. Identification of the CPY sorting receptor offers strong support to the current model of soluble vacuolar protein sorting (Fig. 2). Precursor CPY binds to its receptor in a late-Golgi compartment, effectively removing CPY from the pool Receptor-CPY complexes are of secreted proteins. packaged into transport vesicles and delivered to a prevacuolar endosomal compartment. Here, precursor CPY dissociates from the receptor, possibly because of the acidic environment of the endosomal compartment. The receptor then recycles back to the Golgi for another round of sorting, while precursor CPY moves on to the vacuole where it is matured to its active form. Other vacuolar protein sorting receptors would be expected to function in a similar manner. Many steps outlined in this model would require tight regulation to maintain the efficiency and fidelity of the vacuolar protein sorting system. One class of regulatory molecules that play an essential role in this localization pathway are phosphoinositides.
Roles for a lipid kinase and a protein vacuolar
kinase in
protein sorting
Characterization of the Vpsl5 and Vps34 proteins
The observation that strains deleted for either the I/PSI5 or VPS34 gene share a common set of mutant phenotypes suggests that their gene products function at a similar step in the vacuolar protein sorting pathway These phenotypes include missorting of vacuolar hydrolases, defects in vacuolar segregation during cell division, sensitivity to osmotic stress, the presence of a single enlarged vacuolar compartment, and a temperature-sensitive growth defect [5,8,17-191. Extensive molecular, biochemical and genetic analyses have demonstrated that W’S15 encodes a serinejthreonine protein kinase and I/pS34 encodes a lipid kinase. These proteins act together as part of a key regulatory complex required for vacuolar protein localization (see below;
PW The VPS34 gene encodes a phosphatidylinositol (PtdIns) 3-kinase that shares substantial sequence identity with two cloned mammalian phosphoinositide (PI) 3-kinases [20-231. The yeast enzyme is a PtdIns-specific 3-kinase
0 1995 Current Opinion
in Cell Biolog!
Fig.1.The predicted structure of the CPY sorting receptor, Vpsl Op. Vpsl Op is a type I transmembrane protein (1577 amino acids) containing a 1393 amino acid amino-terminal lumenal domain and a 164 amino acid carboxy-terminal cytoplasmic domain. The lumenal domain contains two regions (domains 1 and 2) that share a 20% sequence identity. Each domain comprises seven -50 amino acid repeat structures that also share significant sequence similarity (numbered l-14). Two cysteine-rich motifs are also present in the lumenal domain (Cys motifs I and II). These motifs comprise 10 cysteine residues; the spacing of the cysteines in each motif is conserved. The functions of these domains and motifs are unknown, but they may be involved in ligand binding. The small cytoplasmic portion of Vpsl Op contains the sequence FYVF (single-letter code for amino acids) which may function as a Colgi retention/retrieval signal required for receptor recycling. The cytoplasmic domain may also interact with other components of the’soning machinery (e.g. Vps35p, Vps29p and/or vesicle coat proteins) that function in the packaging and delivery of the receptor complex to or from the prevacuolar endosomal compartment (see Fig. 2). The shaded box depicts the 20 amino acid transmembrane domain of VpslOp. C, carboxyl terminus; N, amino terminus.
that catalyzes the production only of PtdIns (3) phosphate [PtdIns(3)P] [24*], whereas the mammalian PI 3kinases can catalyze the production of other, more highly phosphorylated phosphoinositides [PtdIns(3,4)Pz and PtdIns(3,4,5)Ps] [25,26]. In addition, the mammalian PI 3-kinases are primarily involved in receptor-mediated signal transduction events at the cell surface and/or receptor trafficking [21,27,28]. In yeast, the Vps34p PtdIns 3-kinase is required for vacuolar protein sorting. Point mutations in highly conserved residues within the lipid kinase catalytic domain of Vps34p result in strains with characteristics indistinguishable from a vps34 null mutant, including vacuolar protein missorting and the absence of detectable cellular PtdIns(3)P [18,20,22]. Recent studies using a vps34ts (temperature-sensitive) strain demonstrate the tight correlation between PtdIns(3)P levels and the efficiency of vacuolar protein sorting [29**]. In the vps34” strain, CPY is sorted to the vacuole in a wild-type manner when the cells are maintained at permissive temperature. Upon shift to the non-permissive temperature (37”C), however, newly synthesized CPY is missorted and secreted. Furthermore, levels of PtdIns(3)P decreased by 80% after a 10min shift to the non-permissive temperature [29**]. This concomitant drop in enzymatic activity and CPY sorting efficiency indicates that the acidic phosphoinositide PtdIns(3)P serves a critical role in vacuolar protein sorting. Interestingly, Vps34p also possess a protein kinase activity; however, the functional role for this activity is not yet known [24-l.
545
546
Membranes and sortine
Fig. 2. A model for vacuolar protein sort-
i
-i
Vpsl P vps15p vps34p Clathrin
/?I,
Sorting
t
VP521 p vps45p Pep
-I ‘1
7
\
Targeting/ /
Endosome Transport vesicle
late Colgi 0 1995
Recently, a mammalian PtdIns 3-kinase that shares physical and biochemical properties with Vps34p has been characterized [30”]. This mammalian PtdIns 3-kinase is relatively insensitive to the drug wortmannin (a PI 3-kinase inhibitor) and shows a PtdIns-specific substrate specificity. These characteristics are similar to those displayed by the yeast Vps34 protein and suggest that this mammalian PtdIns 3-kinase may be involved in protein trafficking events (e.g. lysosomal protein sorting). Characteristics common to Vps34p and the mammalian PtdIns 3-kinases may also be shared by recently cloned plant PI 3-kinases [31*,32’]. Further analysis of these and other PI 3kinases will h.elp to define the function of the different classes of PI 3-kinases found in eukaryotes.
The Vpsl5 protein kinase regulates the Vps34 phosphatidylinositol 3-kinase
The serine/threonine protein kinase Vpsl5p interacts with Vps34p to form a membrane-associated complex localized to the cytoplasmic face of a compartment that most likely corresponds to the late Golgi [20]. Active VpsI5p appears to recruit Vps34p to the membrane: in cells deleted for VPS1.5, Vps34p is no longer associated with cell membranes [20] and its PtdIns 3-kinase activity is dramatically reduced [29**]. Strains carrying point mutations in the I/PSI5 protein kinase domain also show a decrease in Vps34p activity. In addition, inactivation of Vpsl5p in a VpslW strain results in vacuolar protein missorting and in a rapid decrease in cellular PtdIns(3)P levels [29”]. Collectively, these data indicate that the Vpsl5 protein kinase is required to recruit Vps34p to the membrane and activate the enzyme.
Current Opinion in Cell Biology
ing. After transit through the early stages of the yeast secretory pathway, precursor vacuolar proteins (e.g. p2CPY) are sorted away from secretory proteins by receptor molecules (e.g. VpslOp) in a late-Golgi compartment. Receptor-ligand complexes are packaged into transport vesicles and delivered to a prevacuolar endosomal compartment. This leg of the pathway appears to depend on the functions of Vpsl p, Vpsl5p, Vps34p and clathrin. Vps21 p, Vps45p and Pepl2p appear to be required for vesicle targeting and/or fusion. Once at the endosome, the receptor-ligand complexes dissociate and the receptor recycles back to the Colgi for another round of sorting. Vpslp may also be required for this recycling event. Transport of precursor vacuolar proteins from the endosome to the vacuole probably requires the function of Vps33p and Ypt7p. After arrival in the vacuole, the precursors are converted to their mature vacuolar form (e.g. mCPY).
Several possible mechanisms exist by which PtdIns(3)P could regulate protein traffic from the Golgi to the vacuole [20,29**]. First, formation of PtdIns(3)P may act as a signal to recruit or activate specific proteins involved in transport vesicle formation (e.g. coat proteins). Second, PtdIns(3)P may have a role in clustering or segregating vacuolar hydrolase receptor molecules (e.g. VpslOp) for packaging into vesicle carriers. Finally, phosphorylation of membrane PtdIns could initiate vesicle formation by enhancing the curvature of the membrane bilayer at the vesicle bud site due to the increased charge repulsion between phospholipid head groups [33]. Further analysis of vps34 and vps 15 mutants, as well as other vps mutants that affect this stage of the vacuolar protein localization pathway, should help define the role PtdIns(3)P plays in vesicle formation events. Interestingly, a vps34 mutant was recently uncovered in a selection designed to identify strains defective in endocytosis [34*]. Further analysis demonstrated that the peptide mating pheromone a-factor was endocytosed with wild-type kinetics in the vps34 mutant; however, unlike wild-type cells, subsequent vacuolar degradation of the pheromone did not occur. This block in a-factor turnover may be due to defects in endosomal compartment function or a lack of active vacuolar degradative enzymes.
VPSI encodes a dynamin-like
GTPase
The movement of proteins from the Golgi to the vacuole also requires a high molecular weight GTPase encoded by the VPS 1 gene; vps I mutant cells missort and secrete
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
soluble vacuolar proteins [35,36]. Vpslp shows extensive homology with the amino-terminal GTP-binding domain of dynamin [37]. Genetic studies in Drosophila and expression of mutant proteins in mammalian systems have demonstrated that dynamin plays an essential role in endocytosis [38,39]. Recently, dynamin has been shown to localize to the neck of invaginated endocytic vesicles and it probably participates in vesicle fission [40,41]. Vsplp does not appear to play a direct role in the yeast endocytic pathway, but rather has a role in the sorting of vacuolar proteins. The localization of Vpslp to Golgi membranes in yeast [35] and its homology to dynamin suggest a possible role for Vpslp in the formation of Golgi-derived transport vesicles [42*]. However, ups 2 mutations also cause mislocalization of the resident Golgi membrane protein Kex2p; Kex2p is delivered to the vacuole in ~3 1 mutant cells where it is degraded [43,44]. This observation has led to a model in which Vpslp function may be required for retrieving resident Golgi proteins (e.g. Kex2p) as well as the sorting receptors for vacuolar hydrolases (e.g. VpslOp) from the prevacuolar endosomal compartment (Fig. 2). Blocking retrieval of sorting receptors would therefore result in the vacuolar protein sorting defect seen in vpsl mutant cells. A new study [42*] suggests an alternative model for membrane protein sorting and retention in vsl mutant cells that is more consistent with the potential role of Vpslp in vesicle formation at the Golgi. Nothwehr et al. [42*] examined the localization of a fusion protein containing a Golgi retention signal and the vacuolar membrane protein alkaline phosphatase (ALP) in opal mutant strains that also contained sec4 or end4 temperature-sensitive mutations. At permissive temperatures the VP’1 sec4ts or ye l end@s double mutants delivered ALP and the resident Golgi hybrid protein to the vacuole. At non-permissive temperatures, however, the delivery of both proteins was blocked. One explanation for these results is that loss of Vpslp function actually results in the mislocalization of late Golgi and vacuolar membrane proteins to the cell surface, from where they are subsequently delivered to the vacuole via the endocytic pathway. In the double mutants, movement to (se&s) or from (end4ls) the plasma membrane is blocked and the reporter proteins cannot be delivered to the vacuole. In support of this view, these investigators found that in vpsl end4ts double mutants Golgi and vacuolar membrane proteins could be detected on the plasma membrane at the non-permissive temperature by a protease digestion assay [42*]. If Vpslp is involved in the formation of Golgi-derived transport vesicles that deliver membrane and proteins to a prevacuolar endosome, then disruption of this function may result in the diversion of membrane traffic to the plasma membrane [42-l. Further characterization of Vpslp and vpsl mutants will be required to completely differentiate between the vesicle formation and recycling models for Vpslp function. Characterization of a opsl mutant suppressor MVPl may help &rift the role of
Vpslp in membrane
and protein
delivery to the vacuole
[451.
Vesicular trafficking
events in vacuolar
protein
localization Many protein localization systems utilize vesicular carriers to move proteins between subcellular compartments. Genetic and biochemical approaches have been used to identify components that are required for transport vesicle formation, targeting and fusion. These components include small GTP-binding proteins, vesicle coat proteins, NSF, SNAPS, SNARES (SNAP receptors) and members of the Secl protein family (see Bennett, this issue, pp 581-586; [46-481). The conserved function of many of these components has led to a generalized hypothesis for vesicle targeting and fusion [46-48]. In this model, specific proteins on the vesicle carrier (V-SNARES) interact with partner proteins on the target membrane (t-SNARES). The specificity of v-SNARE and t-SNARE pairing results, therefore, in the correct docking of transport vesicles with the appropriate target membrane. Once docked, a generalized vesicle fusion machinery (including SNAPS and NSF) drives membrane fusion between the transport vesicle and target membrane. Small GTP-binding proteins and Seclp homologues are also required in these targeting and/or fusion events.
Small CTP-binding proteins
Members of the GTP-binding, Secl and SNARE protein families have also been found to function in the vacuolar protein sorting system. One of these, Vps2lp, is a small GTP-binding protein of the rab/Ypt/Sec4 family, showing the highest sequence homology to the mammalian rab5 protein [49*,50g]. Mutations within the proposed GTP-binding, effector or prenylation domains of I/ps27 result in a severe vacuolar protein sorting defect [49*]. In addition, vps2l mutant cells accumulate 40-50nm vesicles. These vesicles are probably Golgi-derived transport intermediates that contain precursor vacuolar proteins (Fig. 2). Vesicular intermediates distinct from those seen in vps21 mutant cells also accumulate in yptl and sec4 mutant cells [51,52]. YPTl and SEC4 encode small GTP-binding proteins required for endoplasmic reticulum to Golgi and Golgi to plasma membrane vesicle trafficking events, respectively. The vesicle-accumulation phenotype of the sec4, yptl and vps21 mutants indicates a role for small GTP-binding proteins in vesicle targeting and/or fusion events. In the case of Vps2lp, this GTPase appears to be involved in vesicle targeting and/or fusion with a prevacuolar endosomal compartment [490,5(r] (Fig. 2). Another small GTP-binding protein, Ypt7p, is also required for efficient vacuolar protein sorting. ypr7
547
548
Membranes and sorting
null mutants have perturbed vacuole morphology [53], missort vacuolar hydrolases and are also deficient in the degradation of endocytosed material [53,54]. These phenotypes are consistent with a functional role in the movement of proteins from an endosomal compartment to the vacuole (Fig. 2).
Secl protein homologues Two members of the Secl protein family, Vsp45p and Vsp33p, have also been shown to be required for vacuolar protein sorting [55-581. Like vps21 mutants, vps45 mutants accumulate 40-50nm vesicles and may function in the same Golgi to endosome transport step [55,56]. vps33 mutants lack a vacuole structure and exhibit severe vacuolar protein sorting defects [17]. It is likely that Vps33p functions at a late step in the vacuolar protein sorting pathway, possibly in the delivery of membrane and protein from the endosome to the vacuole. The exact functional role of Secl protein family members remains to be elucidated; however, recent studies in mammalian systems indicated that a neuronal Seclp homologue associates with a t-SNARE, syntaxin, on the presynaptic plasma membrane and may facilitate or stabilize docking of transport vesicles [59-611.
SNARES and vesicle coat proteins
Recently, homology between syntaxin and a gene product involved in vacuolar protein sorting, Pepl2p. has been reported [11,48]. This homology indicates that Pepl2p may function as a t-SNARE in the vacuolar protein sorting system. pep12 mutants (pep12 is alIelic to vps6) missort vacuolar hydrolases and share a number of phenotypes with vps21 and vps45 mutants [5,49*,55,56], suggesting that the three gene products may participate in the same vesicle transport step. To date, no member of the v-SNARE protein family has been identified in the vacuolar protein sorting pathway. Initially, the vesicle coat protein clathrin did not appear to be required for vacuolar protein sorting; cells containing a null allele of the clathrin heavy chain gene, CHCl, did not missort vacuolar proteins [62,63]. This result was surprising given the apparent importance of clathrin function in Golgi to lysosome protein transport in mammalian cells. Only when a temperature-conditional allele of clathrin was characterized did its role in yeast vacuolar protein sorting become apparent [64]. Shortly after a shift to the non-permissive temperature, cells containing a chcl temperature-sensitive allele missorted vacuolar CPY. After prolonged incubations at non-permissive temperatures, however, these cells appeared to adapt to the loss of clathrin function and began to properly sort CPY to the vacuole. The mechanism of this adaptation is presently unclear. No other vesicle coat proteins (e.g. COP1 or COPII) have been shown to participate in Golgi to vacuole transport of vacuolar proteins.
Although it is unclear if other, as yet unidentified, vesicle coat proteins are required in this protein transport system, the identification of small GTP-binding proteins (Vps2lp, Ypt7p), Seclp homologues (Vps45p, Vps33p) and a syntaxin homologue (Pepl2p) indicates that the basic mechanisms used to target, dock and fuse vesicles in the secretory and endocytic pathways are also utilized in the delivery of vacuolar proteins from the Golgi to the endosome and from the endosome to the vacuole.
Vacuole membrane protein localization The vacuolar membrane protein ALP normally transits the early stages of the secretory pathway and is then delivered to the vacuole from the late-Golgi sorting compartment [65]. Earlier studies had indicated that ALP contained vacuolar protein sorting information in its transmembrane or cytoplasmic domains, suggesting that, like soluble vacuolar proteins, the localization of vacuolar membrane proteins was an active process [66]. However, more recent studies using the vacuolar membrane protein dipeptidyl aminopeptidase B (DPAP B) indicate that the delivery of vacuolar membrane proteins may be by a default localization process. By studying the localization of mutant DPAPB molecules or fusion proteins containing various domains of DPAPB fused to reporter proteins, Stevens and co-workers [67] found that vacuolar localization of DPAP B appeared to require only membrane association; no positive vacuolar sorting signals could be found in the lumenal, transmembrane or cytoplasmic domains of DPAPB. In addition, when the retention signals on the resident Golgi proteins Kexlp, Kex2p and Stel3p were mutated, these mutant proteins also traveled to the vacuole [67-701. These observations have lead to the proposal that the vacuole is the default delivery destination for membrane proteins in yeast [71*].
Conclusions Our undemanding of the molecular mechanisms involved in intracellular protein transport has been facilitated by the identification and characterization of the ITS genes and gene products. As more I/I’S genes are uncovered and their gene products characterized, it is becoming apparent that protein delivery to the vacuole is a complex process, involving the highly regulated movement of protein and membrane to and from a number of subcellular compartments. Several conserved functions in vesicle-mediated protein transport systems have been found to play a role in vacuolar protein localization, including small GTP-binding proteins (Vps2lp and Ypt7p), a dynamin homologue (Vpslp), a t-SNARE (Pepl2p) and Secl protein homologues (Vps45p and Vps33p). Further characterization of these proteins and components that interact and/or regulate their
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
function will help dissect the underlying mechanisms of vesicle-mediated protein localization. The continued characterization of the CPY sorting receptor (VpslOp) should help in our understanding of receptor signaling, transport and recycling. Finally, one of the most interesting findings is that a lipid kinase (Vps34p) is essential for the sorting of soluble vacuolar proteins. Defining the contribution and regulation of phospholipid molecules in vesicle-mediated vacuolar protein transport will offer new insights into the general role of lipid modification in protein and membrane trafflcking events throughout the cell.
11.
Jones EW: Proteinase mutants Genetics 1977, 85:23-33.
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cerevisiae.
Stack JH, Horazdovsky BF, Emr SD: Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and CTP-binding proteins. Annu Rev Cell Dev Biol 1995, ll:l-33. This comprehensive review details the roles of a protein kinase, lipid kinase and CTP-binding proteins in vacuolar protein sorting. 12. .
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Stevens TH, Rothman JH, Payne CS, Schekman R: Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. j Cell Biol 1986, 102:1551-1557.
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Rothman JH, Hunter CP, Valls LA, Stevens TH: Overproductioninduced mislocalization of a yeast vacuolar protein allows isolation of its structural gene. Proc Nat/ Acad Sci USA 1986, 83:3248-3252.
15. ..
Acknowledgements We wish to thank the members of the Emr laboratory for many helpful discussions. We especially thank Chris Burd for critically reading this manuscript and especially acknowledge the contribution of Eric Marcusson for uncovering the repeat structures found in VpslOp.
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Stack JH, DeWald DB, Takegawa K, Emr SD: Vesicle-mediated protein transport: regulatory interactions between the VpslS protein kinase and the Vps34 Ptdlns 3-kinase essential for vacuolar protein sorting in yeast. ) Cell Bio/ 1995, 129:321-334. Analysis of vps7Sfirsand vps34rS strains reveal that inactivation of Vpsl 5p or Vps34p results both in a decline of cellular phosphatidylinositol 3-phosphate [Ptdlns(3)P] levels and in the missorting of vacuolar hydrolases. These results imply a direct role for Ptdlns(3)P in protein sorting. The authors also show that kinase-defective forms of Vps34p result in a dominant-negative phenotype whereas kinase-defective forms of VpslSp do not. In addition, chemical cross-linking studies show that Vpsl Sp kinase mutants are defective for association with Vps34p. This indicates that an active Vpsl 5p kinase is required for recruitment of Vps34p to the membrane.
vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with 2 functionally separable domains. ) Cell Biol 1992, 119:773-786. 37.
Obar RA, Collins CA, Hammarback JA, Shpetner HS, Vallee RB: Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of CTP-binding proteins. Nature 1990, 347:25&261.
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Van der Bliek A, Meyerowitz EM: Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 1991, 351:411-414.
40.
Takei K, McPherson PS, Schmid SL, DeCamilli P: Tubular membrane invaginations coated by dynamin rings are induced by GTP-yS in nerve terminals. Name 1995, 374:186-190.
41.
Hinshaw JE, Schmid SL: Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 1995, 374:190-l 92.
29. ..
Stephens L, Cooke FT. Walters R, Jackson T, Volina S, Gout I, Waterfield MD, Hawkins PT: Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol 1994, 4:203-214. A thorough biochemical analysis of a phosphatidylinositol-specific phosphoinositide (PI) 3-kinase from bovine cells. This activity from cytosol was partially purified and has a native molecular weight of 280 kDa. In addition, this mammalian enzyme is resistant to the drug wortmannin, unaffected by adenosine treatment, and not activated by tyrosine-phosphorylated peptides. The authors suggest that this phosphatidylinositol-specific PI 3-kinase may have a fundamentally different role in mammalian cells than the broad-specificity PI 3-kinases. 30. ..
Hong Z, Verma DPS: A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation. Proc Nat/ Acad Sci USA 1994, 91:9617-9621. Two phosphatidylinositol (Ptdlns) 3-kinases from soybean were cloned and characterized. The predicted sequence of these proteins shares approximately 40% sequence identity with yeast Vps34p. Expression of the root form and the nodule form of Ptdlns 3-kinase was documented in different tissues and during nodule organogenesis. 31. .
Welters P, Takegawa K, Emr SD, Chrispeels MJ: AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana is an essential protein with homology to a calcium-dependent lipid binding domain. Proc Nat/ Acad Sci USA 1994, 91:11398-11402. A phosphatidylinositol 3-kinase from Arabidopsis rhaliana was cloned and characterized (40% identity to Vps34p). When antisense constructs of A1Vps34 were expressed at high levels, second generation transgenic A. rhaliana exhibited severe defects in growth and development.
32. .
33.
Sheetz MP, Singer SJ: Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc Nat/ Acad Sci USA 1974, 71:4457-4461.
Munn AL, Riezman H: Endocytosis is required for the growth of vacuolar H+-ATPase-defective yeast: identification of six new end genes. / Cell Viol 1994, 127:373-386. A selection scheme was devised to uncover additional mutants affecting the endocytic pathway. The authors identified six end mutants that are synthetically lethal in combination with vat2 (VAT2 encodes the 60 kDa subunit of the vacuolar H+-ATPase). Two of the end mutants (end72=vps34; end73zvps4) were identified previously as having a role in vacuolar protein sorting. Four new mutants show altered uptake of the a-factor mating pheromone. 34. .
35.
Rothman JH, Raymond CK, Gilbert T, O’Hara PJ, Stevens TH: A putative CTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting. Cell 1990, 61:1063-1074.
36.
Vater CA, Raymond CK, Ekena K, Howaldstevenson I, Stevens TH: The VPSl protein, a homolog of dynamin required for
Nothwehr SF, Conibear E, Stevens TH: Colgi and vacuolar proteins reach the vacuole in vpsl mutant yeast cells via the plasma membrane. / Cell Biol 1995, 129:35-46. _. _ These authors examine the localization ot a late Golgi hybrid membrane protein and a vacuolar membrane protein in vpsl mutant cells that also contain mutations that disrupt membrane flow to or from the plasma membrane (sec4 and end4). The study indicates that resident late-Golgi proteins and vacuolar proteins are delivered to the vacuole via the plasma membrane in vpsl mutant cells. A model is presented in which Vpsl p function is required for the formation of transport vesicles at a late-Colgi compartment. 42. .
43.
Wilsbach K, Payne GS: Vpslp, a member of the dynamin CTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO / 1993, 12:3049-3059.
44.
Wilsbach K, Payne CS: Dynamic retention of TGN membrane proteins in Saccharornyces cerevisiae. Trends Cell Biof 1993, 3:42&432.
45.
Ekena K, Stevens TH: The Saccbaromyces cerevisiae MVPl gene interacts with VPSI and is required for vacuolar protein sorting. MO/ Cell Biol 1995, 15:1671-1678.
46.
Rothman JE, Orci L: Molecular dissection pathway. Nature 1992, 355:409-415.
47.
Rothman JE, Warren G: Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr Biol 1994, 4:220-233.
48.
Bennett MK, Scheller RH: The molecular machinery for secretion is conserved from yeast to neurons. Proc iVar/ Acad Sci USA 1993, 90:2559-2563.
of the secretory
Horazdovsky BF, Busch CR, Emr SD: VPSZI encodes a rab5like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO ) 1994, 13:1297-l 309. This paper describes the charactenzatlon ot a small GTP-binding protein required for vacuolar protein sorting. Mutational analysis demonstrates that conserved GTP-binding, effector and prenylation domains found in Vps2lp are required for function. In addition, small 40-50nm vesicles accumulate in vps27 cells.
49. .
Singer-Kruger B, Stenmark H, Dusterhoft A, Philippsen P, Yoo JS, Gallwitz D, Zerial M: Role of three rabS-like GTPases, YptSlp, Ypt52p, and Yp153p, in the endocytic and vacuolar protein sorting pathways of yeast. ) Cell Biol 1994, 125:283-298. Using a PCR based approach, two homologues of mammalian rab5, YPT.57 and YPT53, were identified in yeast. Another rab5 homologue, YPT52, was found on chromosome XI as part of the yeast genome sequencing initiative. One of these homologues, YPTS7, was found to be the same gene as VPS27. In addition to vacuolar protein sorting defects, ypK7 mutants show a defect in the vacuolar delivery of endocytic markers. These phenotypes were exaggerated in ypdil ypr.52 and ypt57 yptSZypr53 double and triple mutants, indicating a role for
50. .
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr these proteins pathways. 51.
in the late endocytic and vacuolar protein localization
Becker J, Tan TJ, Trepte HH, Callwitz D: Mutational analysis of the putative effector domain of the CTP-binding Yptl protein in yeast suggests specific regulation by a novel GAP activity. EM60 1 1991, 10:785-792.
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Payne GS, Hasson TB, Hasson MS, Schekman R: Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae. MO/ Ce// Biol 1987, 7:3888-3898.
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Seeger M, Payne G: A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EM60 1 1992, 11:2811-2818.
65.
Klionsky DJ, Emr SD: Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EM60 / 1989, 8:2241-2250.
66.
Klionsky DJ, Emr SD: A new class of lysosomal/vacuolar protein sorting signals. / Biol Chem 1990, 2655349-5352.
67.
Roberts CJ, Nothwehr SF, Stevens TH: Membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment. j Cell Biol 1992, 119:69-83.
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Coud B, Salminen A, Walworth N, Novick P: A CTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 1988, 53:753-768.
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Wichmann H, Hen@ L, Callwitz D: Endocytosis in yeast: evidence for the involvement of a small CTP-binding protein (YpBp). Cell 1992, 71:1131-1142.
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Schimmijller F, Riezman H: Involvement of Yp17p, a small CTPase, in trafftc from late endosome to the vacuole in yeast. / Cell Sci 1994, 106:823-830.
55.
Cowles CR, Emr SD, Horazdovsky BF: Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles. ) Cell Sci, 1994, 107:3449-3459.
68.
Wilcox CA, Redding K, Wright R, Fuller RS: Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole. MO/ Bio/ Cell 1992, 3:1353-1371.
56.
Piper RC, Whitters EA, Stevens TH: Yeast Vsp45p is a Seclplike protein required for the consumption of vacuole-targeted, pos&Colgi transport vesicles. Eur 1 Cell Bio/ 1994, 65:305-3 18.
69.
57.
Banta LM, Vida TA, Herman PK, Emr SD: Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. MO/ Cell Biol 1990, 10:4638-4649.
Northwehr SF, Roberts CJ, Stevens TH: Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues. j Cell Bio/ 1993, 121:1197-1209.
70.
Cooper A, Bussey H: Yeast Kexlp is a Go&-associated membrane protein - deletions in a cytoplasmic domain result in the mislocalization to the vacuolar membrane. / Cell 6iol 1992, 119:1459-1468.
58.
Wada Y, Kitamoto K, Kanbe T, Tanaka K, Anraku Y: The SLPI gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function. MO/ Cell Biol 1990, l&2214-2223.
71.
Nothwehr SF, Stevens TH: Sorting of membrane proteins in the yeast secretory pathway. 1 Eio/ Chem 1994, 269: 10185-l 0188. A comprehenslve review that describes vacuolar membrane protein localization, as well as retention and recycling of resident membrane proteins in the secretory and vacuolar protein localization pathways.
.
59.
Hata Y, Slaughter CA, Sijdhof TC: Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 1993, 366:347-351.
60.
Garcia EP, Catti E, Butler M, Burton J, De Camilli P: A rat brain Secl homologue related to Rop and UNC18 interacts with syntaxin. Proc Nat/ Acad Sci USA 1994, 91:2003-2007.
61.
Pevsner J, Shu-Chan H, Scheller RH: n-Secl: a neural-specific syntaxin-binding protein. Proc Nat/ Acad Sci USA 1994, 91:1445-l 449.
BF Horazdovsky, Department of Biochemistry, Texas Southwestern Medical Center, 5323 Harry vard, Dallas, TX 75235-9038, USA.
62.
Payne GS, Baker D, Van Tuinen E, Schekman R: Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast. J Cell No/ 1988, 106:1453-1461.
DB DeWald and SD Emr, Division of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0668, USA.
University of Hines Boule-
551
Daryll B DeWald
Center, Dallas and University
and biochemical
have identified
analyses
Included among these components
of yeast vacuolar
proteins
Some of these gene products are homologous
localization
pathway.
Recent
each of these components offered new insights
Current
kinase, a
and a dynamin-like
to proteins required for
at other stages of the secretory and endocytic pathways.
appear to be required
localization
protein
San Diego, USA
are a sorting receptor, a protein
kinase, small CTP-binding
sorting and transport Others
of California,
more than 40 gene products that play a role in this process.
phosphatidylinositol CTPase.
and Scott D Emr
for unique studies
functions
have helped
plays in vacuolar
into the molecular
Opinion
The vacuole of the yeast Saccharomyces cerevisiae is functionally equivalent to the mammalian lysosome: both are acidic compartments involved primarily in the degradation of macromolecules [1,2]. The degradative nature of the lysosome/vacuole results 6om the presence of a variety of hydrolytic enzymes that have been specifically delivered to these organelles. This localization process is complex, involving a large number of cellular components that function in the recognition and vesicular transport of these hydrolases. Newly synthesized vacuolar hydrolases transit through the early stages of the secretory pathway as inactive zymogens together with proteins that are destined to be secreted from the cell (see Salama and Schekman, this issue, pp 536-543; [3]). Soluble vacuolar hydrolases are actively sorted away from the secretory protein pool in a late-Golgi compartment (possibly equivalent to the trans-Golgi network) and delivered to the vacuole via an endosomal intermediate [4-6]. Upon arrival in the vacuole, the hydrolases are then processed to their mature active forms. In an effort to identify the tram-acting cellular machinery responsible for this schemes have localization process, several selection been developed to identify mutants that mislocalize sorting vacuolar proteins (ups, for vacuolar protein defective) [7-lo]. In addition, mutants that exhibit decreased vacuolar protease activity have been identified (pep, for peptidase deficient) [l 11, many of which also show defects in vacuolar protein delivery [lo]. Together, the ups and pep mutants define more than 40 complementation groups [5], reflecting the complex nature of the vacuolar protein localization pathway.
protein
1995,
protein
the role that
localization
mechanisms
in Cell Biology
Introduction
in the vacuolar to define
and have
of protein sorting.
7:544-551
Recent analyses of the gene products affected in several of the mutants have provided new insights into the mechanisms involved in vacuolar protein localization [12*]; these developments are the subject of this review.
Receptor molecules
for vacuolar
protein sorting
The role of receptor molecules in vacuolar protein localization had been postulated for many years [13]. This proposal was based primarily on the observation that the vacuolar protein sorting system can be saturated. When vacuolar carboxypeptidase Y (CPY) or proteinase A (PrA) were overproduced, a large portion of the overexpressed protein was secreted from the cell in its Golgi-modified precursor form [13,14]. Therefore, it appeared that a limiting component in the vacuolar protein sorting machinery (such as a receptor molecule) was being saturated and the overproduced vacuolar proteins were being redirected to the cell surface by a default localization pathway. Interestingly, the amino-terminal vacuolar protein localization signals of CPY and PrA share no obvious sequence similarity [l], indicating that two different receptor molecules (one for CPY and one for PrA) may be involved in vacuolar protein localization. To identify the gene encoding the proposed CPY sorting receptor, Marcusson et al. [15”] carefully examined the ups mutant collection and uncovered a small group of mutants (vps10, ups.29 and vps3-5) that showed a CPY-specific sorting defect; CPY was secreted in these mutants whereas the vast majority of PrA was correctly delivered to the vacuole [15**, 161. The gene affected in
Abbreviations ALP-alkaline
544
phosphatase; CPY-carboxypeptidase Y; DPAPB-dipeptidyl aminopeptidase 6; PI-phosphoinositide; PrA-proteinase A; Ptdlntphosphatidylinositol; SNARE-SNAP receptor.
0 Current Biology Ltd ISSN 0955-0674
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
one of these mutants, I/PSI@ encodes a CPY sorting receptor. VpslOp is a type I transmembrane protein, with a 1393 amino acid amino-terminal lumenal domain and a cytoplasmic 164 amino acid carboxy-terminal domain (Fig. 1). It is also a stable protein that cofractionates with a late-Golgi marker protein, placing this receptor molecule at the site where vacuolar proteins are sorted. Most importantly, cross-linking experiments show that VpslOp interacts specifically with precursor CPY and this interaction is mediated by the vacuolar sorting signal of CPY [15**]. At present, vps mutant selections have not uncovered a candidate PrA receptor molecule; however, it is likely that other vacuolar protein sorting receptors are present in this pathway. Identification of the CPY sorting receptor offers strong support to the current model of soluble vacuolar protein sorting (Fig. 2). Precursor CPY binds to its receptor in a late-Golgi compartment, effectively removing CPY from the pool Receptor-CPY complexes are of secreted proteins. packaged into transport vesicles and delivered to a prevacuolar endosomal compartment. Here, precursor CPY dissociates from the receptor, possibly because of the acidic environment of the endosomal compartment. The receptor then recycles back to the Golgi for another round of sorting, while precursor CPY moves on to the vacuole where it is matured to its active form. Other vacuolar protein sorting receptors would be expected to function in a similar manner. Many steps outlined in this model would require tight regulation to maintain the efficiency and fidelity of the vacuolar protein sorting system. One class of regulatory molecules that play an essential role in this localization pathway are phosphoinositides.
Roles for a lipid kinase and a protein vacuolar
kinase in
protein sorting
Characterization of the Vpsl5 and Vps34 proteins
The observation that strains deleted for either the I/PSI5 or VPS34 gene share a common set of mutant phenotypes suggests that their gene products function at a similar step in the vacuolar protein sorting pathway These phenotypes include missorting of vacuolar hydrolases, defects in vacuolar segregation during cell division, sensitivity to osmotic stress, the presence of a single enlarged vacuolar compartment, and a temperature-sensitive growth defect [5,8,17-191. Extensive molecular, biochemical and genetic analyses have demonstrated that W’S15 encodes a serinejthreonine protein kinase and I/pS34 encodes a lipid kinase. These proteins act together as part of a key regulatory complex required for vacuolar protein localization (see below;
PW The VPS34 gene encodes a phosphatidylinositol (PtdIns) 3-kinase that shares substantial sequence identity with two cloned mammalian phosphoinositide (PI) 3-kinases [20-231. The yeast enzyme is a PtdIns-specific 3-kinase
0 1995 Current Opinion
in Cell Biolog!
Fig.1.The predicted structure of the CPY sorting receptor, Vpsl Op. Vpsl Op is a type I transmembrane protein (1577 amino acids) containing a 1393 amino acid amino-terminal lumenal domain and a 164 amino acid carboxy-terminal cytoplasmic domain. The lumenal domain contains two regions (domains 1 and 2) that share a 20% sequence identity. Each domain comprises seven -50 amino acid repeat structures that also share significant sequence similarity (numbered l-14). Two cysteine-rich motifs are also present in the lumenal domain (Cys motifs I and II). These motifs comprise 10 cysteine residues; the spacing of the cysteines in each motif is conserved. The functions of these domains and motifs are unknown, but they may be involved in ligand binding. The small cytoplasmic portion of Vpsl Op contains the sequence FYVF (single-letter code for amino acids) which may function as a Colgi retention/retrieval signal required for receptor recycling. The cytoplasmic domain may also interact with other components of the’soning machinery (e.g. Vps35p, Vps29p and/or vesicle coat proteins) that function in the packaging and delivery of the receptor complex to or from the prevacuolar endosomal compartment (see Fig. 2). The shaded box depicts the 20 amino acid transmembrane domain of VpslOp. C, carboxyl terminus; N, amino terminus.
that catalyzes the production only of PtdIns (3) phosphate [PtdIns(3)P] [24*], whereas the mammalian PI 3kinases can catalyze the production of other, more highly phosphorylated phosphoinositides [PtdIns(3,4)Pz and PtdIns(3,4,5)Ps] [25,26]. In addition, the mammalian PI 3-kinases are primarily involved in receptor-mediated signal transduction events at the cell surface and/or receptor trafficking [21,27,28]. In yeast, the Vps34p PtdIns 3-kinase is required for vacuolar protein sorting. Point mutations in highly conserved residues within the lipid kinase catalytic domain of Vps34p result in strains with characteristics indistinguishable from a vps34 null mutant, including vacuolar protein missorting and the absence of detectable cellular PtdIns(3)P [18,20,22]. Recent studies using a vps34ts (temperature-sensitive) strain demonstrate the tight correlation between PtdIns(3)P levels and the efficiency of vacuolar protein sorting [29**]. In the vps34” strain, CPY is sorted to the vacuole in a wild-type manner when the cells are maintained at permissive temperature. Upon shift to the non-permissive temperature (37”C), however, newly synthesized CPY is missorted and secreted. Furthermore, levels of PtdIns(3)P decreased by 80% after a 10min shift to the non-permissive temperature [29**]. This concomitant drop in enzymatic activity and CPY sorting efficiency indicates that the acidic phosphoinositide PtdIns(3)P serves a critical role in vacuolar protein sorting. Interestingly, Vps34p also possess a protein kinase activity; however, the functional role for this activity is not yet known [24-l.
545
546
Membranes and sortine
Fig. 2. A model for vacuolar protein sort-
i
-i
Vpsl P vps15p vps34p Clathrin
/?I,
Sorting
t
VP521 p vps45p Pep
-I ‘1
7
\
Targeting/ /
Endosome Transport vesicle
late Colgi 0 1995
Recently, a mammalian PtdIns 3-kinase that shares physical and biochemical properties with Vps34p has been characterized [30”]. This mammalian PtdIns 3-kinase is relatively insensitive to the drug wortmannin (a PI 3-kinase inhibitor) and shows a PtdIns-specific substrate specificity. These characteristics are similar to those displayed by the yeast Vps34 protein and suggest that this mammalian PtdIns 3-kinase may be involved in protein trafficking events (e.g. lysosomal protein sorting). Characteristics common to Vps34p and the mammalian PtdIns 3-kinases may also be shared by recently cloned plant PI 3-kinases [31*,32’]. Further analysis of these and other PI 3kinases will h.elp to define the function of the different classes of PI 3-kinases found in eukaryotes.
The Vpsl5 protein kinase regulates the Vps34 phosphatidylinositol 3-kinase
The serine/threonine protein kinase Vpsl5p interacts with Vps34p to form a membrane-associated complex localized to the cytoplasmic face of a compartment that most likely corresponds to the late Golgi [20]. Active VpsI5p appears to recruit Vps34p to the membrane: in cells deleted for VPS1.5, Vps34p is no longer associated with cell membranes [20] and its PtdIns 3-kinase activity is dramatically reduced [29**]. Strains carrying point mutations in the I/PSI5 protein kinase domain also show a decrease in Vps34p activity. In addition, inactivation of Vpsl5p in a VpslW strain results in vacuolar protein missorting and in a rapid decrease in cellular PtdIns(3)P levels [29”]. Collectively, these data indicate that the Vpsl5 protein kinase is required to recruit Vps34p to the membrane and activate the enzyme.
Current Opinion in Cell Biology
ing. After transit through the early stages of the yeast secretory pathway, precursor vacuolar proteins (e.g. p2CPY) are sorted away from secretory proteins by receptor molecules (e.g. VpslOp) in a late-Golgi compartment. Receptor-ligand complexes are packaged into transport vesicles and delivered to a prevacuolar endosomal compartment. This leg of the pathway appears to depend on the functions of Vpsl p, Vpsl5p, Vps34p and clathrin. Vps21 p, Vps45p and Pepl2p appear to be required for vesicle targeting and/or fusion. Once at the endosome, the receptor-ligand complexes dissociate and the receptor recycles back to the Colgi for another round of sorting. Vpslp may also be required for this recycling event. Transport of precursor vacuolar proteins from the endosome to the vacuole probably requires the function of Vps33p and Ypt7p. After arrival in the vacuole, the precursors are converted to their mature vacuolar form (e.g. mCPY).
Several possible mechanisms exist by which PtdIns(3)P could regulate protein traffic from the Golgi to the vacuole [20,29**]. First, formation of PtdIns(3)P may act as a signal to recruit or activate specific proteins involved in transport vesicle formation (e.g. coat proteins). Second, PtdIns(3)P may have a role in clustering or segregating vacuolar hydrolase receptor molecules (e.g. VpslOp) for packaging into vesicle carriers. Finally, phosphorylation of membrane PtdIns could initiate vesicle formation by enhancing the curvature of the membrane bilayer at the vesicle bud site due to the increased charge repulsion between phospholipid head groups [33]. Further analysis of vps34 and vps 15 mutants, as well as other vps mutants that affect this stage of the vacuolar protein localization pathway, should help define the role PtdIns(3)P plays in vesicle formation events. Interestingly, a vps34 mutant was recently uncovered in a selection designed to identify strains defective in endocytosis [34*]. Further analysis demonstrated that the peptide mating pheromone a-factor was endocytosed with wild-type kinetics in the vps34 mutant; however, unlike wild-type cells, subsequent vacuolar degradation of the pheromone did not occur. This block in a-factor turnover may be due to defects in endosomal compartment function or a lack of active vacuolar degradative enzymes.
VPSI encodes a dynamin-like
GTPase
The movement of proteins from the Golgi to the vacuole also requires a high molecular weight GTPase encoded by the VPS 1 gene; vps I mutant cells missort and secrete
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
soluble vacuolar proteins [35,36]. Vpslp shows extensive homology with the amino-terminal GTP-binding domain of dynamin [37]. Genetic studies in Drosophila and expression of mutant proteins in mammalian systems have demonstrated that dynamin plays an essential role in endocytosis [38,39]. Recently, dynamin has been shown to localize to the neck of invaginated endocytic vesicles and it probably participates in vesicle fission [40,41]. Vsplp does not appear to play a direct role in the yeast endocytic pathway, but rather has a role in the sorting of vacuolar proteins. The localization of Vpslp to Golgi membranes in yeast [35] and its homology to dynamin suggest a possible role for Vpslp in the formation of Golgi-derived transport vesicles [42*]. However, ups 2 mutations also cause mislocalization of the resident Golgi membrane protein Kex2p; Kex2p is delivered to the vacuole in ~3 1 mutant cells where it is degraded [43,44]. This observation has led to a model in which Vpslp function may be required for retrieving resident Golgi proteins (e.g. Kex2p) as well as the sorting receptors for vacuolar hydrolases (e.g. VpslOp) from the prevacuolar endosomal compartment (Fig. 2). Blocking retrieval of sorting receptors would therefore result in the vacuolar protein sorting defect seen in vpsl mutant cells. A new study [42*] suggests an alternative model for membrane protein sorting and retention in vsl mutant cells that is more consistent with the potential role of Vpslp in vesicle formation at the Golgi. Nothwehr et al. [42*] examined the localization of a fusion protein containing a Golgi retention signal and the vacuolar membrane protein alkaline phosphatase (ALP) in opal mutant strains that also contained sec4 or end4 temperature-sensitive mutations. At permissive temperatures the VP’1 sec4ts or ye l end@s double mutants delivered ALP and the resident Golgi hybrid protein to the vacuole. At non-permissive temperatures, however, the delivery of both proteins was blocked. One explanation for these results is that loss of Vpslp function actually results in the mislocalization of late Golgi and vacuolar membrane proteins to the cell surface, from where they are subsequently delivered to the vacuole via the endocytic pathway. In the double mutants, movement to (se&s) or from (end4ls) the plasma membrane is blocked and the reporter proteins cannot be delivered to the vacuole. In support of this view, these investigators found that in vpsl end4ts double mutants Golgi and vacuolar membrane proteins could be detected on the plasma membrane at the non-permissive temperature by a protease digestion assay [42*]. If Vpslp is involved in the formation of Golgi-derived transport vesicles that deliver membrane and proteins to a prevacuolar endosome, then disruption of this function may result in the diversion of membrane traffic to the plasma membrane [42-l. Further characterization of Vpslp and vpsl mutants will be required to completely differentiate between the vesicle formation and recycling models for Vpslp function. Characterization of a opsl mutant suppressor MVPl may help &rift the role of
Vpslp in membrane
and protein
delivery to the vacuole
[451.
Vesicular trafficking
events in vacuolar
protein
localization Many protein localization systems utilize vesicular carriers to move proteins between subcellular compartments. Genetic and biochemical approaches have been used to identify components that are required for transport vesicle formation, targeting and fusion. These components include small GTP-binding proteins, vesicle coat proteins, NSF, SNAPS, SNARES (SNAP receptors) and members of the Secl protein family (see Bennett, this issue, pp 581-586; [46-481). The conserved function of many of these components has led to a generalized hypothesis for vesicle targeting and fusion [46-48]. In this model, specific proteins on the vesicle carrier (V-SNARES) interact with partner proteins on the target membrane (t-SNARES). The specificity of v-SNARE and t-SNARE pairing results, therefore, in the correct docking of transport vesicles with the appropriate target membrane. Once docked, a generalized vesicle fusion machinery (including SNAPS and NSF) drives membrane fusion between the transport vesicle and target membrane. Small GTP-binding proteins and Seclp homologues are also required in these targeting and/or fusion events.
Small CTP-binding proteins
Members of the GTP-binding, Secl and SNARE protein families have also been found to function in the vacuolar protein sorting system. One of these, Vps2lp, is a small GTP-binding protein of the rab/Ypt/Sec4 family, showing the highest sequence homology to the mammalian rab5 protein [49*,50g]. Mutations within the proposed GTP-binding, effector or prenylation domains of I/ps27 result in a severe vacuolar protein sorting defect [49*]. In addition, vps2l mutant cells accumulate 40-50nm vesicles. These vesicles are probably Golgi-derived transport intermediates that contain precursor vacuolar proteins (Fig. 2). Vesicular intermediates distinct from those seen in vps21 mutant cells also accumulate in yptl and sec4 mutant cells [51,52]. YPTl and SEC4 encode small GTP-binding proteins required for endoplasmic reticulum to Golgi and Golgi to plasma membrane vesicle trafficking events, respectively. The vesicle-accumulation phenotype of the sec4, yptl and vps21 mutants indicates a role for small GTP-binding proteins in vesicle targeting and/or fusion events. In the case of Vps2lp, this GTPase appears to be involved in vesicle targeting and/or fusion with a prevacuolar endosomal compartment [490,5(r] (Fig. 2). Another small GTP-binding protein, Ypt7p, is also required for efficient vacuolar protein sorting. ypr7
547
548
Membranes and sorting
null mutants have perturbed vacuole morphology [53], missort vacuolar hydrolases and are also deficient in the degradation of endocytosed material [53,54]. These phenotypes are consistent with a functional role in the movement of proteins from an endosomal compartment to the vacuole (Fig. 2).
Secl protein homologues Two members of the Secl protein family, Vsp45p and Vsp33p, have also been shown to be required for vacuolar protein sorting [55-581. Like vps21 mutants, vps45 mutants accumulate 40-50nm vesicles and may function in the same Golgi to endosome transport step [55,56]. vps33 mutants lack a vacuole structure and exhibit severe vacuolar protein sorting defects [17]. It is likely that Vps33p functions at a late step in the vacuolar protein sorting pathway, possibly in the delivery of membrane and protein from the endosome to the vacuole. The exact functional role of Secl protein family members remains to be elucidated; however, recent studies in mammalian systems indicated that a neuronal Seclp homologue associates with a t-SNARE, syntaxin, on the presynaptic plasma membrane and may facilitate or stabilize docking of transport vesicles [59-611.
SNARES and vesicle coat proteins
Recently, homology between syntaxin and a gene product involved in vacuolar protein sorting, Pepl2p. has been reported [11,48]. This homology indicates that Pepl2p may function as a t-SNARE in the vacuolar protein sorting system. pep12 mutants (pep12 is alIelic to vps6) missort vacuolar hydrolases and share a number of phenotypes with vps21 and vps45 mutants [5,49*,55,56], suggesting that the three gene products may participate in the same vesicle transport step. To date, no member of the v-SNARE protein family has been identified in the vacuolar protein sorting pathway. Initially, the vesicle coat protein clathrin did not appear to be required for vacuolar protein sorting; cells containing a null allele of the clathrin heavy chain gene, CHCl, did not missort vacuolar proteins [62,63]. This result was surprising given the apparent importance of clathrin function in Golgi to lysosome protein transport in mammalian cells. Only when a temperature-conditional allele of clathrin was characterized did its role in yeast vacuolar protein sorting become apparent [64]. Shortly after a shift to the non-permissive temperature, cells containing a chcl temperature-sensitive allele missorted vacuolar CPY. After prolonged incubations at non-permissive temperatures, however, these cells appeared to adapt to the loss of clathrin function and began to properly sort CPY to the vacuole. The mechanism of this adaptation is presently unclear. No other vesicle coat proteins (e.g. COP1 or COPII) have been shown to participate in Golgi to vacuole transport of vacuolar proteins.
Although it is unclear if other, as yet unidentified, vesicle coat proteins are required in this protein transport system, the identification of small GTP-binding proteins (Vps2lp, Ypt7p), Seclp homologues (Vps45p, Vps33p) and a syntaxin homologue (Pepl2p) indicates that the basic mechanisms used to target, dock and fuse vesicles in the secretory and endocytic pathways are also utilized in the delivery of vacuolar proteins from the Golgi to the endosome and from the endosome to the vacuole.
Vacuole membrane protein localization The vacuolar membrane protein ALP normally transits the early stages of the secretory pathway and is then delivered to the vacuole from the late-Golgi sorting compartment [65]. Earlier studies had indicated that ALP contained vacuolar protein sorting information in its transmembrane or cytoplasmic domains, suggesting that, like soluble vacuolar proteins, the localization of vacuolar membrane proteins was an active process [66]. However, more recent studies using the vacuolar membrane protein dipeptidyl aminopeptidase B (DPAP B) indicate that the delivery of vacuolar membrane proteins may be by a default localization process. By studying the localization of mutant DPAPB molecules or fusion proteins containing various domains of DPAPB fused to reporter proteins, Stevens and co-workers [67] found that vacuolar localization of DPAP B appeared to require only membrane association; no positive vacuolar sorting signals could be found in the lumenal, transmembrane or cytoplasmic domains of DPAPB. In addition, when the retention signals on the resident Golgi proteins Kexlp, Kex2p and Stel3p were mutated, these mutant proteins also traveled to the vacuole [67-701. These observations have lead to the proposal that the vacuole is the default delivery destination for membrane proteins in yeast [71*].
Conclusions Our undemanding of the molecular mechanisms involved in intracellular protein transport has been facilitated by the identification and characterization of the ITS genes and gene products. As more I/I’S genes are uncovered and their gene products characterized, it is becoming apparent that protein delivery to the vacuole is a complex process, involving the highly regulated movement of protein and membrane to and from a number of subcellular compartments. Several conserved functions in vesicle-mediated protein transport systems have been found to play a role in vacuolar protein localization, including small GTP-binding proteins (Vps2lp and Ypt7p), a dynamin homologue (Vpslp), a t-SNARE (Pepl2p) and Secl protein homologues (Vps45p and Vps33p). Further characterization of these proteins and components that interact and/or regulate their
Protein transport to the yeast vacuole Horazdovsky, DeWald and Emr
function will help dissect the underlying mechanisms of vesicle-mediated protein localization. The continued characterization of the CPY sorting receptor (VpslOp) should help in our understanding of receptor signaling, transport and recycling. Finally, one of the most interesting findings is that a lipid kinase (Vps34p) is essential for the sorting of soluble vacuolar proteins. Defining the contribution and regulation of phospholipid molecules in vesicle-mediated vacuolar protein transport will offer new insights into the general role of lipid modification in protein and membrane trafflcking events throughout the cell.
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15. ..
Acknowledgements We wish to thank the members of the Emr laboratory for many helpful discussions. We especially thank Chris Burd for critically reading this manuscript and especially acknowledge the contribution of Eric Marcusson for uncovering the repeat structures found in VpslOp.
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University of Hines Boule-
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