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.     

The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function

Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.     

A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn2+ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.     

Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast

Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans. The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.     

Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities

The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40-50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases.     

Is NADH effective in the treatment of Parkinson''s disease?

SNAREs are compartmentally specific membrane proteins required for intracellular membrane fusion. Homologues of the Saccharomyces cerevisiae protein Sec1p interact with, and are likely to be involved in regulation of, the syntaxin family of SNAREs. In yeast there are 7 functionally distinct syntaxins but only four clearly identifiable homologues of Sec1p. One of these, Vps45p, is required for transport from Golgi to late endosomes, and has been implicated in the function of the late endosomal syntaxin Pep12p. However, there is evidence that not all the functions of Pep12p are equally dependent on Vps45p, and conversely that the phenotypes of vps45 mutants cannot be explained entirely by loss of Pep12p activity. We have recently characterised two yeast syntaxins which function in trans-Golgi or endosomal compartments, Tlg1p and Tlg2p. We show here that the principal binding site for Vps45p on intracellular membranes is provided by Tlg2p rather than Pep12p, and that Vps45p is required for stable expression of Tlg2p. Vps45p is also associated with Tlg1p as part of a triple complex containing both Tlg1p and Tlg2p. Since a deltavps45 deltatlg2 double mutant has a more severe vacuolar protein sorting defect than a deltatlg2 mutant, Vps45p cannot only interact with Tlg2p. It appears that the role of Vps45p in protein traffic is more complex than has previously been assumed.     

Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body

Sorting of signal-transducing cell surface receptors within multivesicular bodies (MVBs) is required for their rapid down-regulation and degradation within lysosomes. Yeast mutants defective in late stages of transport to the vacuole/lysosome accumulate MVBs. We demonstrate that the membrane glycoprotein carboxypeptidase S and the G protein-coupled receptor Ste2p are targeted into the vacuole lumen, and this process requires a subset of VPS gene products essential for normal endosome function. The PtdIns(3)P 5-kinase activity of Fab1p, which converts the product of the Vps34p PtdIns 3-kinase PtdIns(3)P into PtdIns(3,5)P2, also is required for cargo-selective sorting into the vacuole lumen. These findings demonstrate a role for phosphoinositide signaling at distinct stages of vacuolar/lysosomal protein transport and couple PtdIns(3,5)P2 synthesis to regulation of MVB sorting.     

Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.     

Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast

pep12/vps6 mutants of Saccharomyces cerevisiae are defective in delivery of soluble vacuolar hydrolases to the vacuole. Morphological analysis by electron microscopy revealed that pep12 cells accumulate 40- to 50-nm vesicles. Furthermore, pep12 cells have enlarged vacuoles characteristic of class D pep/vps mutants. PEP12 encodes a protein of 288 amino acids that has a C-terminal hydrophobic region and shares significant sequence similarity with members of the syntaxin protein family. These proteins appear to participate in the docking and fusion of intracellular transport vesicles. Pep12p is the first member of the syntaxin family to be implicated in transport between the Golgi and the vacuole/lysosome. Pep12p-specific polyclonal antisera detected a 35-kDa protein that fractionated as an integral membrane protein. Subcellular fractionation experiments revealed that Pep12p was associated with membrane fractions of two different densities; the major pool (approximately 90%) of pep12p may associate with the endosome, while a minor pool (approximately 10%) cofractionated with the late Golgi marker Kex2p. These observations suggest that Pep12p may mediate the docking of Golgi-derived transport vesicles at the endosome.     

Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.     

A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast

We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G. Marcusson, J.-L. Cereghino, and S.D. Emr. 1997. J. Cell Biol. 137:79-92). By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p. This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p. Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction. Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles. We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding. Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation. Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation. Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.     

Characterization of a novel yeast SNARE protein implicated in Golgi retrograde traffic

The protein trafficking machinery of eukaryotic cells is employed for protein secretion and for the localization of resident proteins of the exocytic and endocytic pathways. Protein transit between organelles is mediated by transport vesicles that bear integral membrane proteins (v-SNAREs) which selectively interact with similar proteins on the target membrane (t-SNAREs), resulting in a docked vesicle. A novel Saccharomyces cerevisiae SNARE protein, which has been termed Vti1p, was identified by its sequence similarity to known SNAREs. Vti1p is a predominantly Golgi-localized 25-kDa type II integral membrane protein that is essential for yeast viability. Vti1p can bind Sec17p (yeast SNAP) and enter into a Sec18p (NSF)-sensitive complex with the cis-Golgi t-SNARE Sed5p. This Sed5p/Vti1p complex is distinct from the previously described Sed5p/Sec22p anterograde vesicle docking complex. Depletion of Vti1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the Golgi. Temperature-sensitive mutants of Vti1p show a similar carboxypeptidase Y trafficking defect, but the secretion of invertase and gp400/hsp150 is not significantly affected. The temperature-sensitive vti1 growth defect can be rescued by the overexpression of the v-SNARE, Ykt6p, which physically interacts with Vti1p. We propose that Vti1p, along with Ykt6p and perhaps Sft1p, acts as a retrograde v-SNARE capable of interacting with the cis-Golgi t-SNARE Sed5p.     

Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.     

Expression of rat cathepsin D cDNA in Saccharomyces cerevisiae: implications for intracellular targeting of cathepsin D to vacuoles

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.     

PAT1, a microtubule-interacting protein, recognizes the basolateral sorting signal of amyloid precursor protein

In epithelial cells, sorting of membrane proteins to the basolateral surface depends on the presence of a basolateral sorting signal (BaSS) in their cytoplasmic domain. Amyloid precursor protein (APP), a basolateral protein implicated in the pathogenesis of Alzheimer's disease, contains a tyrosine-based BaSS, and mutation of the tyrosine residue results in nonpolarized transport of APP. Here we report identification of a protein, termed PAT1 (protein interacting with APP tail 1), that interacts with the APP-BaSS but binds poorly when the critical tyrosine is mutated and does not bind the tyrosine-based endocytic signal of APP. PAT1 shows homology to kinesin light chain, which is a component of the plus-end directed microtubule-based motor involved in transporting membrane proteins to the basolateral surface. PAT1, a cytoplasmic protein, associates with membranes, cofractionates with APP-containing vesicles, and binds microtubules in a nucleotide-sensitive manner. Cotransfection of PAT1 with a reporter protein shows that PAT1 is functionally linked with intracellular transport of APP. We propose that PAT1 is involved in the translocation of APP along microtubules toward the cell surface.     

The vesicle transport protein Vps33p is an ATP-binding protein that localizes to the cytosol in an energy-dependent manner

Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole. Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20-40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of approximately 40 min. Depletion of cellular ATP increased the association rate to a half-time of approximately 4 min and caused 80-90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. The N-ethylmaleimide-sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy-dependent sedimentation characteristics as Vps33p. We determined that the photosensitive analog, 8-azido-[alpha-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[alpha-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole. After depleting cellular ATP, Vps33p-GFP appeared to localize with a punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.     

A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole

Transport of yeast alkaline phosphatase (ALP) to the vacuole depends on the clathrin adaptor-like complex AP-3, but does not depend on proteins necessary for transport through pre-vacuolar endosomes. We have identified ALP sequences that direct sorting into the AP-3-dependent pathway using chimeric proteins containing residues from the ALP cytoplasmic domain fused to sequences from a Golgi-localized membrane protein, guanosine diphosphatase (GDPase). The full-length ALP cytoplasmic domain, or ALP amino acids 1-16 separated from the transmembrane domain by a spacer, directed GDPase chimeric proteins from the Golgi complex to the vacuole via the AP-3 pathway. Mutation of residues Leu13 and Val14 within the ALP cytoplasmic domain prevented AP-3-dependent vacuolar transport of both chimeric proteins and full-length ALP. This Leucine-Valine (LV)-based sorting signal targeted chimeric proteins and native ALP to the vacuole in cells lacking clathrin function. These results identify an LV-based sorting signal in the ALP cytoplasmic domain that directs transport into a clathrin-independent, AP-3-dependent pathway to the vacuole. The similarity of the ALP sorting signal to mammalian dileucine sorting motifs, and the evolutionary conservation of AP-3 subunits, suggests that dileucine-like signals constitute a core element for AP-3-dependent transport to lysosomal compartments in all eukaryotic cells.     

Sec35p, a novel peripheral membrane protein, is required for ER to Golgi vesicle docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (. Genetics. 142:393-406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.     

Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.