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.     

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.     

Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, T. gondii possesses several plant‐like features, such as the chloroplast‐like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant‐like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L‐like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca²⁺/H⁺ and Na⁺/H⁺ exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca²⁺ store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers colocalizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well.     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

Characterization of<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis> mutants defective in vacuolar acidification and protein sorting

The vacuolar H⁺-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca²⁺. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1 ⁺ or vma3 ⁺ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe.Communicated by M. Johnston     

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.     

Proton pumps of the vacuolar membrane in growing plant cells

Plant growth results from the division, enlargement and specialization of cells. The two processes of the enlargement and the differentiation of cells are not spatially separated in plant tissue. We focus our attention here on the enlargement and elongation of cells. In most cases, growing plant cells contain a large central vacuole. The acid growth theory is based on the space-filling function of the large vacuole. The active transport systems in the vacuolar membrane are essential for maintenance of high osmotic pressure and for the expansion of the vacuole. The secondary active transport systems of the vacuole for sugars and ions are driven by the proton-motive force which is generated by the vacuolar H⁺-ATPase and H⁺-translocating inorganic pyrophosphatase. In this review, the relationship between cell elongation and these enzymes of the vacuolar membrane is emphasized.     

Mechanistic differences in the uptake of salicylic acid glucose conjugates by vacuolar membrane‐enriched vesicles isolated from Arabidopsis thaliana

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2‐O‐β‐d ‐glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane‐enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A₁ (vacuolar H⁺‐ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H⁺‐antiporter. Despite its absence in the vacuole, we observed the MgATP‐dependent uptake of SGE by Arabidopsis vacuolar membrane‐enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A₁ and gramicidin D providing evidence that uptake was dependent on an H⁺‐antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP‐dependent SAG and SGE uptake exhibited Michaelis–Menten‐type saturation kinetics. The vacuolar enriched‐membrane vesicles had a 46‐fold greater affinity and a 10‐fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long‐term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations.     

Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4⁺ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA₄‐tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1⁺, ESCRT‐0 hse1⁺ or ESCRT‐I sst6⁺ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin‐treated cells, Shu1‐Nbr1 and Shu1‐Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin‐treated cells harboring a deletion for one of the following genes: ubi4⁺, nbr1⁺, hse1⁺ and sst6⁺. Together, these results reveal that hemin‐mediated vacuolar targeting of Shu1 requires Ubi4‐dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.     

Endosomal functions in plants

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins.     

TRANSPARENT TESTA 13 is a tonoplast P3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P‐type H⁺‐ATPases in the plasma membrane, and multimeric vacuolar‐type H⁺‐ATPases (V‐ATPases) and vacuolar H⁺‐pyrophosphatases (H⁺‐PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P_3A‐ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA‐filled central vacuoles as observed in the wild‐type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P‐type H⁺‐ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H⁺‐PPase VHP1. Our findings indicate that the P_3A‐ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12‐mediated transport of PA precursors to the vacuole.     

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX‐type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na⁺ in vacuoles. However, all isoforms characterized so far catalyze both Na⁺/H⁺ and K⁺/H⁺ exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K⁺, which in turn affects plant K⁺ nutrition and Na⁺ tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K⁺ vacuolar pools in all growth conditions tested, but no consistent enhancement of Na⁺ accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K⁺ and to withstand salt‐shock. Under K⁺‐limiting conditions, greater K⁺ compartmentation in the vacuole occurred at the expense of the cytosolic K⁺ pool, which was lower in transgenic plants. This caused the early activation of the high‐affinity K⁺ uptake system, enhanced K⁺ uptake by roots, and increased the K⁺ content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H⁺‐linked K⁺ transport that is thought to facilitate active K⁺ uptake at the tonoplast, and the partitioning of K⁺ between vacuole and cytosol.     

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.     

V H-ATPase along the yeast secretory pathway: Energization of the ER and Golgi membranes

H⁺ transport driven by V H⁺-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca²⁺-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H⁺ transport activity of P H⁺-ATPase by more than 75% while that of V H⁺-ATPase remained unchanged. ER H⁺ ATPase exhibits higher resistance to bafilomycin (I₅₀ = 38.4 nM) than Golgi and vacuole pumps (I₅₀ = 0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H⁺ transport mediated by V H⁺-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H⁺-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

A Novel Plant Vacuolar Na+/H+ Antiporter Gene Evolved by DNA Shuffling Confers Improved Salt Tolerance in Yeast

Plant vacuolar Na⁺/H⁺ antiporters play important roles in maintaining cellular ion homeostasis and mediating the transport of Na⁺ out of the cytosol and into the vacuole. Vacuolar antiporters have been shown to play significant roles in salt tolerance; however the relatively low V_max of the Na⁺/H⁺ exchange of the Na⁺/H⁺ antiporters identified could limit its application in the molecular breeding of salt tolerant crops. In this study, we applied DNA shuffling methodology to generate and recombine the mutations of Arabidopsis thaliana vacuolar Na⁺/H⁺ antiporter gene AtNHX1. Screening using a large scale yeast complementation system identified AtNHXS1, a novel Na⁺/H⁺ antiporter. Expression of AtNHXS1 in yeast showed that the antiporter localized to the vacuolar membrane and that its expression improved the tolerance of yeast to NaCl, KCl, LiCl, and hygromycin B. Measurements of the ion transport activity across the intact yeast vacuole demonstrated that the AtNHXS1 protein showed higher Na⁺/H⁺ exchange activity and a slightly improved K⁺/H⁺ exchange activity.     

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.     

Inorganic phosphate uptake in intact vacuoles isolated from suspension-cultured cells of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don under varying Pi status

Inorganic phosphate (Pi) uptake across the vacuolar membrane of intact vacuoles isolated from Catharanthus roseus suspension-cultured cells was measured. Under low Pi status, Pi uptake into the vacuole was strongly activated compared to high Pi status. Since Pi uptake across the vacuolar membrane is correlated with H⁺ pumping, we examined the dependency of H⁺ pumping on plant Pi status. Both H⁺ pumping and the activities of the vacuolar H⁺-pumps, the V-type H⁺-ATPase and the H⁺-PPase were enhanced under low Pi status. Despite this increase in H⁺ pumping, Western blot analysis showed no distinct increase in the amount of proton pump proteins. Possible mechanisms for the activation of Pi uptake into the vacuole under low Pi status are discussed. Miwa Ohnishi and Tetsuro Mimura contributed equally to this work.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast

Although COPI function on the early secretory pathway in eukaryotes is well established, earlier studies also proposed a nonconventional role for this coat complex in endocytosis in mammalian cells. Here we present results that suggest an involvement for specific COPI subunits in the late steps of endosomal protein sorting in Saccharomyces cerevisiae. First, we found that carboxypeptidase Y (CPY) was partially missorted to the cell surface in certain mutants of the COPIB subcomplex (COPIb; Sec27, Sec28, and possibly Sec33), which indicates an impairment in endosomal transport. Second, integral membrane proteins destined for the vacuolar lumen (i.e., carboxypeptidase S [CPS1]; Fur4, Ste2, and Ste3) accumulated at an aberrant late endosomal compartment in these mutants. The observed phenotypes for COPIb mutants resemble those of class E vacuolar protein sorting (vps) mutants that are impaired in multivesicular body (MVB) protein sorting and biogenesis. Third, we observed physical interactions and colocalization between COPIb subunits and an MVB-associated protein, Vps27. Together, our findings suggest that certain COPI subunits could have a direct role in vacuolar protein sorting to the MVB compartment.