An ER membrane protein,Sop4, facilitates ER export of the yeast plasma membrane [H+]ATPase,Pma1

We have analyzed the mechanism by which Sop4, a novel ER membrane protein, regulates quality control and intracellular transport of Pma1–7, a mutant plasma membrane ATPase. At the restrictive temperature, newly synthesized Pma1–7 is targeted for vacuolar degradation instead of being correctly delivered to the cell surface. Loss of Sop4 at least partially corrects vacuolar mislocalization, allowing Pma1–7 routing to the plasma membrane. Ste2–3 is a mutant pheromone receptor which, like Pma1–7, is defective in targeting to the cell surface, resulting in a mating defect. sop4Δ suppresses the mating defect of ste2–3 cells as well as the growth defect of pma1–7 . Visualization of newly synthesized Pma1–7 in sop4Δ cells by indirect immunofluorescence reveals delayed export from the ER. Similarly, ER export of wild-type Pma1 is delayed in the absence of Sop4 although intracellular transport of Gas1 and CPY is unaffected. These observations suggest a model in which a selective increase in ER residence time for Pma1–7 may allow it to achieve a more favorable conformation for subsequent delivery to the plasma membrane. In support of this model, newly synthesized Pma1–7 is also routed to the plasma membrane upon release from a general block of ER-to-Golgi transport in sec13–1 cells.     

Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1^ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.     

Ubiquitin-mediated targeting of a mutant plasma membrane ATPase, Pma1-7, to the endosomal/vacuolar system in yeast

Pma1-7 is a mutant plasma membrane ATPase that is impaired in targeting to the cell surface at 37 degrees C and is delivered instead to the endosomal/vacuolar pathway for degradation. We have proposed that Pma1-7 is a substrate for a Golgibased quality control mechanism. By contrast with wild-type Pma1, Pma1-7 is ubiquitinated. Ubiquitination and endosomal targeting of Pma1-7 is dependent on the Rsp5-Bul1-Bul2 ubiquitin ligase protein complex but not the transmembrane ubiquitin ligase Tul1. Analysis of Pma1-7 ubiquitination in mutants blocked in protein transport at various steps of the secretory pathway suggests that ubiquitination occurs after ER exit but before endosomal entry. In the absence of ubiquitination in rsp5-1 cells, Pma1-7 is delivered to the cell surface and remains stable. Nevertheless, Pma1-7 remains impaired in association with detergent-insoluble glycolipid-enriched complexes in rsp5-1 cells, suggesting that ubiquitination is not the cause of Pma1-7 exclusion from rafts. In vps1 cells in which protein transport into the endosomal pathway is blocked, Pma1-7 is routed to the cell surface. On arrival at the plasma membrane in vps1 cells, Pma1-7 remains stable and its ubiquitination disappears, suggesting deubiquitination activity at the cell surface. We suggest that Pma1-7 sorting and fate are regulated by ubiquitination.     

Yeast mutants affecting possible quality control of plasma membrane proteins

Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1(ts)). These suppressors inhibited the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 [M. Babst and S. Emr, personal communication] and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with green fluorescent protein [GFP]) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen.     

Golgi and vacuolar membrane proteins reach the vacuole in vps1 mutant yeast cells via the plasma membrane

The Vps1 protein of Saccharomyces cerevisiae is an 80-kD GTPase associated with the Golgi apparatus. Vps1p appears to play a direct role in the retention of late Golgi membrane proteins, which are mislocalized to the vacuolar membrane in its absence. The pathway by which late Golgi and vacuolar membrane proteins reach the vacuole in vps1 delta mutants was investigated by analyzing transport of these proteins in vps1 delta cells that also contained temperature sensitive mutations in either the SEC4 or END4 genes, which are required for a late step in secretion and the internalization step of endocytosis, respectively. Not only was vacuolar transport of a Golgi membrane protein blocked in the vps1 delta sec4-ts and vps1 delta end4-ts double mutant cells at the non-permissive temperature but vacuolar delivery of the vacuolar membrane protein, alkaline phosphatase was also blocked in these cells. Moreover, both proteins expressed in the vps1 delta end4- ts cells at the elevated temperature could be detected on the plasma membrane by a protease digestion assay indicating that these proteins are transported to the vacuole via the plasma membrane in vps1 mutant cells. These data strongly suggest that a loss of Vps1p function causes all membrane traffic departing from the late Golgi normally destined for the prevacuolar compartment to instead be diverted to the plasma membrane. We propose a model in which Vps1p is required for formation of vesicles from the late Golgi apparatus that carry vacuolar and Golgi membrane proteins bound for the prevacuolar compartment.     

Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole

We have characterized a class of mutations in PMA1, (encoding plasma membrane ATPase) that is ideal for the analysis of membrane targeting in Saccharomyces cerevisiae. This class of pma1 mutants undergoes growth arrest at the restrictive temperature because newly synthesized ATPase fails to be targeted to the cell surface. Instead, mutant ATPase is delivered to the vacuole, where it is degraded. Delivery to the vacuole occurs without previous arrival at the plasma membrane because degradation of mutant ATPase is not prevented when internalization from the cell surface is blocked. Disruption of PEP4, encoding vacuolar proteinase A, blocks ATPase degradation, but fails to restore growth because the ATPase is still improperly targeted. One of these pma1 mutants was used to select multicopy suppressors that would permit growth at the nonpermissive temperature. A novel gene, AST1, identified by this selection, suppresses several pma1 alleles defective for targeting. The basis for suppression is that multicopy AST1 causes rerouting of mutant ATPase from the vacuole to the cell surface. pma1 mutants deleted for AST1 have a synthetic growth defect at the permissive temperature, providing genetic evidence for interaction between AST1 and PMA1. Ast1 is a cytoplasmic protein that associates with membranes, and is localized to multiple compartments, including the plasma membrane. The identification of AST1 homologues suggests that Ast1 belongs to a novel family of proteins that participates in membrane traffic.     

Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.

More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.     

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.     

pH-dependent cargo sorting from the Golgi

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.     

The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.     

Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast

The proton pumping H+-ATPase, Pma1, is one of the most abundant integral membrane proteins of the yeast plasma membrane. Pma1 activity controls the intracellular pH and maintains the electrochemical gradient across the plasma membrane, two essential cellular functions. The maintenance of the proton gradient, on the other hand, also requires a specialized lipid composition of this membrane. The plasma membrane of eukaryotic cells is typically rich in sphingolipids and sterols. These two lipids condense to form less fluid membrane microdomains or lipid rafts. The yeast sphingolipid is peculiar in that it invariably contains a saturated very long-chain fatty acid with 26 carbon atoms. During cell growth and plasma membrane expansion, both C26-containing sphingolipids and Pma1 are first synthesized in the endoplasmatic reticulum from where they are transported by the secretory pathway to the cell surface. Remarkably, shortening the C26 fatty acid to a C22 fatty acid by mutations in the fatty acid elongation complex impairs raft association of newly synthesized Pma1 and induces rapid degradation of the ATPase by rerouting the enzyme from the plasma membrane to the vacuole, the fungal equivalent of the lysosome. Here, we review the role of lipids in mediating raft association and stable surface transport of the newly synthesized ATPase, and discuss a model, in which the newly synthesized ATPase assembles into a membrane environment that is enriched in C26-containing lipids already in the endoplasmatic reticulum. The resulting protein-lipid complex is then transported and sorted as an entity to the plasma membrane. Failure to successfully assemble this lipid-protein complex results in mistargeting of the protein to the vacuole.     

Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast

The proton pumping H(+)-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Delta mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414-4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid "rafts"). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.     

Eps1, a novel PDI-related protein involved in ER quality control in yeast.

PMA1 is an essential gene encoding the yeast plasma membrane [H(+)]ATPase. A pma1-D378N mutant has a dominant-negative effect on cell growth because both newly synthesized mutant and wild-type Pma1 molecules are retained and degraded in the endoplasmic reticulum (ER). Like other substrates for ER-associated degradation, Pma1-D378N is stabilized in mutants defective in components of the ubiquitination machinery. A genetic selection was performed for eps (ER-retained pma1 suppressing) mutants in which the growth defect caused by the D378N allele is suppressed. In an eps1 mutant, both mutant and wild-type Pma1 molecules are allowed to travel to the plasma membrane; however, normal retention of resident ER proteins Shr3 and Kar2 is not perturbed. Eps1 is a novel membrane protein belonging to the protein disulfide isomerase (PDI) family, and Eps1 co-localizes with Pma1-D378N in the ER. In the absence of Pma1-D378N, ER export of wild-type Pma1 is not affected by eps1 deletion, but export of the plasma membrane protein Gas1 is delayed. Because Eps1 is required for retention and degradation of Pma1-D378N, we propose a model in which Eps1 acts as a novel membrane-bound chaperone in ER quality control.     

A Mutant Plasma Membrane Protein Is Stabilized Upon Loss of Yvh1, a Novel Ribosome Assembly Factor

Pma1-10 is a mutant plasma membrane ATPase defective at the restrictive temperature in stability at the cell surface. At 37°, Pma1-10 is ubiquitinated and internalized from the plasma membrane for degradation in the vacuole. YVH1, encoding a tyrosine phosphatase, is a mutant suppressor of pma1-10; in the absence of Yvh1, Pma1-10 remains stable at the plasma membrane, thereby permitting cells to grow. The RING finger domain of Yvh1, but not its phosphatase domain, is required for removal of mutant Pma1-10 from the plasma membrane. Yvh1 is a novel ribosome assembly factor: in yvh1Δ cells, free 60S and 80S ribosomal subunits are decreased, free 40S subunits are increased, and half-mer polysomes are accumulated. Pma1-10 is also stabilized by deletion of 60S ribosomal proteins Rpl19a and Rpl35a. We propose that changes in ribosome biogenesis caused by loss of Yvh1 or specific ribosomal proteins have effects on the plasma membrane, perhaps by producing specific translational changes.     

Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.     

The sodium pump Ena1p provides mechanistic insight into the salt sensitivity of vacuolar protein sorting mutants

The vacuolar/endosomal network has an important but as yet undefined role in the cellular tolerance to salt stress. We hypothesized that the mechanistic basis for the importance of vacuolar protein sorting (vps) components in salt tolerance is the targeting of the crucial sodium exporter Ena1p to the plasma membrane. The link between Ena1p and the vps components was established by the observation that overexpression of Ena1p could suppress the salt sensitivity of the ESCRT knockouts vps20Delta, snf7/vps32Delta and snf8/vps22Delta. To further investigate this functional interaction, fluorescence microscopy was utilized to monitor localization of GFP-tagged Ena1p. For all analyzed vps mutants, Ena1p seemed properly localized to the plasma membrane, even during saline growth. However, quantitative differences in plasma membrane localized Ena1p were recorded; e.g. the highly salt sensitive pep12Delta mutant exhibited substantially enhanced Ena1p levels. In addition, the kinetics of Ena1p localization to the plasma membrane was severely delayed in several vps mutants, and this delay correlated to the salt specific growth defect. This paper discusses potential mechanistic hypotheses, like Ena1p transporter activity or localization kinetics, or ESCRT component's influence on signaling, for linking endosomal sorting functions to cellular salt sensitivity.     

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.