Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells

We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; alpha-, gamma-, and delta-TIP) and yellow fluorescent protein (YFP). We also produced soluble reporters consisting of the monomeric red fluorescent protein (RFP) and either the C-terminal vacuolar sorting signal of phaseolin or the sequence-specific sorting signal of proricin. In transgenic Arabidopsis leaves, mature roots, and root tips, all TIP fusions localized to the tonoplast of the central vacuole and both of the lumenal RFP reporters were found within TIP-delimited vacuoles. In embryos from developing, mature, and germinating seeds, all three TIPs localized to the tonoplast of protein storage vacuoles. To determine the temporal TIP expression patterns and to rule out mistargeting due to overexpression, we generated plants expressing YFP fused to the complete genomic sequences of the three TIP isoforms. In transgenic Arabidopsis, gamma-TIP expression was limited to vegetative tissues, but specifically excluded from root tips, whereas alpha-TIP was exclusively expressed during seed maturation. delta-TIP was expressed in vegetative tissues, but not root tips, at a later stage than gamma-TIP. Our findings indicate that, in the Arabidopsis tissues analyzed, two different vacuolar sorting signals target soluble proteins to a single vacuolar location. Moreover, TIP isoform distribution is tissue and development specific, rather than organelle specific.     

Lack of a vacuolar sorting receptor leads to non-specific missorting of soluble vacuolar proteins in Arabidopsis seeds

The plant vacuolar sorting receptor (VSR) binds proteins carrying vacuolar sorting signals (VSS) of the 'sequence-specific' type (ssVSS) but not the C-terminal, hydrophobic sorting signals (ctVSS). Seeds of Arabidopsis mutants lacking the major VSR isoform, AtVSR1, secrete a proportion of the proteins destined to storage vacuoles. The sorting signals for these proteins are not well defined, but they do not seem to be of the ssVSS type. Here, we tested whether absence of VSR1 in seeds leads to secretion of reporter proteins carrying ssVSS but not ctVSS. Our results show that reporters carrying either ssVSS or ctVSS are equally secreted in the absence of VSR1. We discuss our findings in relation to the current model for vacuolar sorting.     

Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.     

Evaluating heavy metal contents in nine composts using four digestion methods

The production and application of compost potentially contaminate the environment with heavy metals. The heavy metal contents of composts must be exactly determined. Therefore, this study aims to understand the heavy metal contents in composts, using different digestion methods and to recommend the most appropriate digestion method making this measurement. Nine composts from different sources, including swine manure, poultry litter, food waste, municipal sewage sludge, soybean meal, wood residues, flesh-bone dust, fish-bone meal and guano, were selected to be digested by four methods to determine the contents of Cd, Cr, Cu, Mn, Ni, Pb and Zn by AAS. The four digestion methods were nitric acid, dry ashing, nitric-perchloric acid and sulfuric acid methods. Analytical results indicated that the nitric acid procedure was the most efficient for recovering Cd, Mn and Ni from most compost samples. The sulfuric acid procedure yielded the lowest recovery of Pb from the certified reference material (BCR 146) and the compost samples, especially from the composts with high pH and EC values potentially high in Ca. The nitric acid procedure was recommended as the method for digesting the composts herein, based on recovery analysis, cost and time taken. Dry ashing was recommended as a flexible method. Nitric-perchloric acid procedure was not recommended because perchloric acid is potentially hazardous during digestion and it recovers relatively little heavy metal.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast.

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.     

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.     

Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV.     

Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.     

The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.     

Acidification of the lysosome-like vacuole and the vacuolar H+-ATPase are deficient in two yeast mutants that fail to sort vacuolar proteins

Organelle acidification plays a demonstrable role in intracellular protein processing, transport, and sorting in animal cells. We investigated the relationship between acidification and protein sorting in yeast by treating yeast cells with ammonium chloride and found that this lysosomotropic agent caused the mislocalization of a substantial fraction of the newly synthesized vacuolar (lysosomal) enzyme proteinase A (PrA) to the cell surface. We have also determined that a subset of the vpl mutants, which are deficient in sorting of vacuolar proteins (Rothman, J. H., and T. H. Stevens. 1986. Cell. 47:1041-1051; Rothman, J. H., I. Howald, and T. H. Stevens. EMBO [Eur. Mol. Biol. Organ.] J. In press), failed to accumulate the lysosomotropic fluorescent dye quinacrine within their vacuoles, mimicking the phenotype of wild-type cells treated with ammonium. The acidification defect of vpl3 and vpl6 mutants correlated with a marked deficiency in vacuolar ATPase activity, diminished levels of two immunoreactive subunits of the protontranslocating ATPase (H+-ATPase) in purified vacuolar membranes, and accumulation of the intracellular portion of PrA as the precursor species. Therefore, some of the VPL genes are required for the normal function of the yeast vacuolar H+-ATPase complex and may encode either subunits of the enzyme or components required for its assembly and targeting. Collectively, these findings implicate a critical role for acidification in vacuolar protein sorting and zymogen activation in yeast, and suggest that components of the yeast vacuolar acidification system may be identified by examining mutants defective in sorting of vacuolar proteins.     

Phenotypic changes in Arabidopsis caused by expression of a yeast vacuolar Ca2+/H+ antiporter

In plants, cytosolic Ca²⁺ levels are tightly regulated, and changes in cytosolic Ca²⁺ have been implicated in converting numerous signals into adapted responses. Vacuolar ion transporters are thought to be key mediators of cytosolic Ca²⁺ concentrations. In an attempt to interpret the role of vacuolar Ca²⁺ transport in plant processes, we have expressed the yeast vacuolar Ca²⁺/H⁺ antiporter, VCX1, in Arabidopsis and tobacco. This transporter localizes to the plant vacuolar membrane. VCX1-expressing Arabidopsis plants displayed increased sensitivity to sodium and other ions. These ion sensitivities could be suppressed by addition of calcium to the media. VCX1-expressing plants demonstrated increased tonoplast-enriched Ca²⁺/H⁺ antiport activity as well as increased Ca²⁺ accumulation. These results suggest that VCX1 expression in Arabidopsis could be a valuable tool with which to experimentally dissect the role of Ca²⁺ transport around the plant vacuole.     

Mutants impaired in vacuolar metal mobilization identify chloroplasts as a target for cadmium hypersensitivity in Arabidopsis thaliana

Cadmium (Cd) is highly toxic to plants causing growth reduction and chlorosis. It binds thiols and competes with essential transition metals. It affects major biochemical processes such as photosynthesis and the redox balance, but the connection between cadmium effects at the biochemical level and its deleterious effect on growth has seldom been established. In this study, two Cd hypersensitive mutants, cad1‐3 impaired in phytochelatin synthase (PCS1), and nramp3nramp4 impaired in release of vacuolar metal stores, have been compared. The analysis combines genetics with measurements of photosynthetic and antioxidant functions. Loss of AtNRAMP3 and AtNRAMP4 function or of PCS1 function leads to comparable Cd sensitivity. Root Cd hypersensitivities conferred by cad1‐3 and nramp3nramp4 are cumulative. The two mutants contrast in their tolerance to oxidative stress. In nramp3nramp4, the photosynthetic apparatus is severely affected by Cd, whereas it is much less affected in cad1‐3. In agreement with chloroplast being a prime target for Cd toxicity in nramp3nramp4, the Cd hypersensitivity of this mutant is alleviated in the dark. The Cd hypersensitivity of nramp3nramp4 mutant highlights the critical role of vacuolar metal stores to supply essential metals to plastids and maintain photosynthetic function under Cd and oxidative stresses.     

Control of cellular physiology by TM9 proteins in yeast and Dictyostelium

TM9 proteins constitute a well defined family, characterized by the presence of a large variable extracellular domain and nine putative transmembrane domains. This family is highly conserved throughout evolution and comprises three members in Dictyostelium discoideum and Saccharomyces cerevisiae and four in humans and mice. In Dictyostelium, previous analysis demonstrated that TM9 proteins are implicated in cellular adhesion. In this study, we generated TM9 mutants in S. cerevisiae and analyzed their phenotype with particular attention to cellular adhesion. S. cerevisiae strains lacking any one of the three TM9 proteins were severely suppressed for adhesive growth and filamentous growth under conditions of nitrogen starvation. In these mutants, expression of the FLO11-lacZ reporter gene was strongly reduced, whereas expression of FRE(Ty1)-lacZ was not, suggesting that TM9 proteins are implicated at a late stage of nutrient-controlled signaling pathways. We also reexamined the phenotype of Dictyostelium TM9 mutant cells, focusing on nutrient-controlled cellular functions. Although the initiation of multicellular development and autophagy was unaltered in Dictyostelium TM9 mutants, nutrient-controlled secretion of lysosomal enzymes was dysregulated in these cells. These results suggest that in both yeast and amoebae, TM9 proteins participate in the control of specific cellular functions in response to changing nutrient conditions.     

The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast

Several lines of evidence suggest that regulation of intracellular Ca(2+) levels is crucial for adaptation of plants to environmental stress. We have cloned and characterized Arabidopsis auto-inhibited Ca(2+)-ATPase, isoform 4 (ACA4), a calmodulin-regulated Ca(2+)-ATPase. Confocal laser scanning data of a green fluorescent protein-tagged version of ACA4 as well as western-blot analysis of microsomal fractions obtained from two-phase partitioning and Suc density gradient centrifugation suggest that ACA4 is localized to small vacuoles. The N terminus of ACA4 contains an auto-inhibitory domain with a binding site for calmodulin as demonstrated through calmodulin-binding studies and complementation experiments using the calcium transport yeast mutant K616. ACA4 and PMC1, the yeast vacuolar Ca(2+)-ATPase, conferred protection against osmotic stress such as high NaCl, KCl, and mannitol when expressed in the K616 strain. An N-terminally modified form of ACA4 specifically conferred increased NaCl tolerance, whereas full-length ATPase had less effect.     

Functional characterization of selected LEA proteins from Arabidopsis thaliana in yeast and in vitro

Main conclusion

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are “atypical”, since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.     

A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast.

We have investigated the role of clathrin in vacuolar protein sorting using yeast strains harboring a temperature-sensitive allele of clathrin heavy chain (chc1-ts). After a 5 min incubation at the non-permissive temperature (37 degrees C), the chc1-ts strains displayed a severe defect in the sorting of lumenal vacuolar proteins. Sorting of a vacuolar membrane protein, alkaline phosphatase, and transport to the surface of a cell wall protein, was not affected at 37 degrees C. In chc1-ts cells incubated at 37 degrees C, secretion of the missorted lumenal vacuolar protein carboxypeptidase Y (CPY) was blocked by the sec1 mutation which prevents fusion of secretory vesicles to the plasma membrane. Unexpectedly, chc1-ts cells incubated for extended periods at 37 degrees C regained the ability to sort CPY. Cells carrying deletions of the CHC1 gene (chc1 delta) also sorted CPY to the vacuole even when subjected to temperature shifts. Vacuolar delivery of CPY in chc1 delta cells was not blocked by sec1 suggesting that transport does not occur by secretion and endocytosis. These results provide in vivo evidence that clathrin plays a role in the Golgi complex in sorting of vacuolar proteins from the secretory pathway. With time, however, yeast cells lacking functional clathrin heavy chains are able to adapt in a way that allows restoration of vacuolar protein sorting in the Golgi complex. These conclusions clarify previous studies of chc1 delta cells which raised the possibility that clathrin is not involved in vacuolar protein sorting.     

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.     

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.