Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting.     

env1 Mutant of VPS35 gene exhibits unique protein localization and processing phenotype at Golgi and lysosomal vacuole in Saccharomyces cerevisiae

The yeast vacuole is functionally and structurally equivalent to the mammalian lysosome. Delivery of resident and cargo proteins to the lysosome is vital for proper cellular operations, and failure to correctly target proteins to the organelle is correlated with the development of neurodegenerative and lysosomal storage diseases. We previously reported a novel mutant screen for vacuolar trafficking defects in yeast Saccharomyces cerevisiae that resulted in the isolation of env1, an allelic mutant of VPS35. As a member of the retromer complex, Vps35p binds directly to cargos and facilitates their retrograde transport to trans Golgi from endosomes. Our previous studies established that env1 exhibits unique pleiotropic phenotype in comparison to other tested VPS35 alleles including severe growth sensitivity to hygromycin B and internal accumulation of the precursor form of the vacuolar enzyme carboxypeptidase Y. Here, through a combination of sub-cellular fractionation and indirect immunofluorescence microscopy, we confirm and extend the unique phenotype of env1 to processing and localization of additional proteins within the vacuolar trafficking pathway. In comparative studies with a null and an allelic mutant of VPS35, env1 exhibited unique processing defects of retromer-independent vacuolar membrane enzyme alkaline phosphatase at the vacuole and significant Golgi localization of retromer cargos Vps10p and Kex2p despite compromised trafficking at the Golgi and late endosome interface.     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

Vps10p cycles between the TGN and the late endosome via the plasma membrane in clathrin mutants

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in a chc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport in chc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, in chc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normal VPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.     

Yeast Vps55p,a functional homolog of human obesity receptor gene-related protein,is involved in late endosome to vacuole trafficking

The Saccharomyces cerevisiae VPS55 (YJR044c) gene encodes a small protein of 140 amino acids with four potential transmembrane domains. VPS55 belongs to a family of genes of unknown function, including the human gene encoding the obesity receptor gene-related protein (OB-RGRP). Yeast cells with a disrupted VPS55 present normal vacuolar morphology, but exhibit an abnormal secretion of the Golgi form of the soluble vacuolar carboxypeptidase Y. However, trafficking of the membrane-bound vacuolar alkaline phosphatase remains normal. The endocytosis of uracil permease, used as an endocytic marker, is normal in vps55Delta cells, but its degradation is delayed and this marker transiently accumulates in late endosomal compartments. We also found that Vps55p is mainly localized in the late endosomes. Collectively, these results indicate that Vps55p is involved in late endosome to vacuole trafficking. Finally, we show that human OB-RGRP displays the same distribution as Vps55p and corrects the phenotypic defects of the vps55Delta strain. Therefore, the function of Vps55p has been conserved throughout evolution. This study highlights the importance of the multispanning Vps55p and OB-RGRP in membrane trafficking to the vacuole/lysosome of eukaryotic cells.     

Pep12p is a multifunctional yeast syntaxin that controls entry of biosynthetic, endocytic and retrograde traffic into the prevacuolar compartment

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p. Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole. Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC. The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC. Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC. Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27 -independent fashion.     

Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae

Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.     

A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.     

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.     

Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.     

A proteomic analysis of secretory proteins of a pre-vacuolar mutant of Candida albicans

S. cerevisiae mutants lacking VPS4 missort several vacuolar proteins to the extracellular space, including carboxypeptidase (CPY), vacuolar protease A (PrA), and vacuolar protease B (PrB). In addition, certain soluble secretory proteins, such as invertase and acid phosphatase, are missorted from the pre-vacuolar compartment (PVC) to the general secretory pathway prior to exocytosis. Although little is known about sorting of proteins via the PVC in Candida albicans, we have previously demonstrated that the C. albicans vps4Δ null mutant missorts PrA and CPY extracellularly, but fails to secrete the aspartyl proteases Sap2p and Sap4–6p. To further define the role of C. albicans VPS4 in the trafficking of pre-vacuolar proteins, we have used 2 dimensional gel electrophoresis (2-DE) and mass spectrometry techniques to study soluble proteins in the supernatants of planktonic cultures obtained from the C. albicans vps4Δ mutant compared to control strain DAY185. Results indicated that lack of VPS4 results in a decrease of canonically secreted proteins whilst having a limited effect on non-canonically secreted extracellular proteins. Four canonically secreted proteins (Cht3p, Pra1p, Mp65p and Sun41p) were identified as reduced in the supernatants from the mutant strain. We also indentified two other major consequences of lack of VPS4, likely associated with secretion defects: altered branching and biofilm formation.     

Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.     

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.     

VPS21 controls entry of endocytosed and biosynthetic proteins into the yeast prevacuolar compartment

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.     

VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins.

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.     

Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p.

In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome.     

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis.

vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.     

PXA Domain-Containing Protein Pxa1 Is Required for Normal Vacuole Function and Morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.     

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Arabidopsis VPS18 plays an important role in regulating pollen tube growth through mediating the late endocytic trafficking and secretion of pectin and associated enzymes to the cell wall.