Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.     

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.     

The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP-1 complex: evidence for functional interaction with clathrin at the Golgi complex.

Clathrin-associated protein (AP) complexes have been implicated in the assembly of clathrin coats and the selectivity of clathrin-mediated protein transport processes. We have identified a yeast gene, APS1, encoding a homolog of the small (referred to herein as sigma) subunits of the mammalian AP-1 complex. Sequence comparisons have shown that Aps1p is more similar to the sigma subunit of the Golgi-localized mammalian AP-1 complex than Aps2p, which is more related to the plasma membrane AP-2 sigma subunit. Like their mammalian counterparts, Aps1p and Aps2p are components of distinct, large (> 200 kDa) complexes and a significant portion of the Aps proteins co-fractionate with clathrin-coated vesicles during gel filtration chromatography. Unexpectedly, even though the evolutionary conservation of AP small subunits is substantial (50% identity between mammalian and yeast proteins), disruptions of APS1 (aps1 delta) and APS2 (aps2 delta), individually or in combination, elicit no detectable mutant phenotypes. These data indicate that the Aps proteins are not absolutely required for clathrin-mediated selective protein transport in cells expressing wild type clathrin. However, aps1 delta accentuated the slow growth and alpha-factor pheromone maturation defect of cells carrying a temperature-sensitive allele of clathrin heavy chain (Chc) (chc1-ts). In contrast, aps1 delta did not influence the effects of chc1-ts on vacuolar protein sorting or receptor-mediated endocytosis. The aps2 delta mutation resulted in a slight effect on chc1-ts cell growth but had no additional effects. The growth defect of cells completely lacking Chc was compounded by aps1 delta but not aps2 delta. These results comprise evidence that Aps1p is involved in a subset of clathrin functions at the Golgi apparatus. The effect of aps1 delta on cells devoid of clathrin function suggests that Aps1p also participates in clathrin-independent processes.     

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.     

Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the organelles and cellular functions involved in transport of the vacuolar glycoprotein, carboxypeptidase Y (CPY). Others have shown that CPY (61 kd) is synthesized as an inactive proenzyme (69 kd) that is matured by cleavage of an 8 kd amino-terminal propeptide. sec mutants that are blocked in either of two early stages in the secretory process and accumulate endoplasmic reticulum or Golgi bodies also accumulate precursor forms of CPY when cells are incubated at the nonpermissive temperature (37°C). These forms are converted to a proper size when cells are returned to a permissive temperature (25°C). Vacuoles isolated from sec mutant cells do not contain the proCPY produced at 37°C. These results suggest that vacuolar and secretory glycoproteins require the same cellular functions for transport from the endoplasmic reticulum and from the Golgi body. The Golgi body represents a branch point in the pathway: from this organelle, vacuolar proenzymes are transported to the vacuole for proteolytic processing and secretory proteins are packaged into vesicles.     

Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway

We have devised a genetic selection for mutant yeast cells that fail to properly deliver the vacuolar glycoprotein CPY to the lysosome-like vacuole. This has allowed us to identify mutations in eight VPL complementation groups that result in aberrant secretion of up to approximately 90% of the immunoreactive CPY. Other soluble vacuolar proteins are also affected by each vpl mutation, demonstrating that a sorting system for multiple vacuolar proteins exists in yeast. Mislocalized CPY apparently traverses late stages of the secretory pathway, since a vesicle-accumulating sec1 mutation prevents secretion of this protein. Despite the presence of abnormal membrane-enclosed organelles in some of the vpl mutants, maturation and secretion of invertase are not substantially perturbed. Thus vpl mutations define a new class of genes that encode products required for sorting of newly synthesized vacuolar proteins from secretory proteins during their transit through the yeast secretory pathway.     

Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment

We are studying intercompartmental protein transport to the yeast lysosome-like vacuole with a reconstitution assay using permeabilized spheroplasts that measures, in an ATP and cytosol dependent reaction, vacuolar delivery and proteolytic maturation of the Golgi-modified precursor forms of vacuolar hydrolases like carboxypeptidase Y (CPY). To identify the potential donor compartment in this assay, we used subcellular fractionation procedures that have uncovered a novel membrane-enclosed prevacuolar transport intermediate. Differential centrifugation was used to separate permeabilized spheroplasts into 15K and 150K g membrane pellets. Centrifugation of these pellets to equilibrium on sucrose density gradients separated vacuolar and Golgi complex marker enzymes into light and dense fractions, respectively. When the Golgi-modified precursor form of CPY (p2CPY) was examined (after a 5-min pulse, 30-s chase), as much as 30-40% fractionated with an intermediate density between both the vacuole and the Golgi complex. Pulse-chase labeling and fractionation of membranes indicated that p2CPY in this gradient region had already passed through the Golgi complex, which kinetically ordered it between the Golgi and the vacuole. A mutant CPY protein that lacks a functional vacuolar sorting signal was detected in Golgi fractions but not in the intermediate compartment indicating that this corresponds to a post-sorting compartment. Based on the low transport efficiency of the mutant CPY protein in vitro (decreased by sevenfold), this intermediate organelle most likely represents the donor compartment in our reconstitution assay. This organelle is not likely to be a transport vesicle intermediate because EM analysis indicates enrichment of 250-400 nm compartments and internalization of surface-bound 35S-alpha-factor at 15 degrees C resulted in its apparent cofractionation with wild-type p2CPY, indicating an endosome-like compartment (Singer, B., and H. Reizman. 1990. J. Cell Biol. 110:1911-1922). Fractionation of p2CPY accumulated in the temperature sensitive vps15 mutant revealed that the vps15 transport block did not occur in the endosome-like compartment but rather in the late Golgi complex, presumably the site of CPY sorting. Therefore, as seen in mammalian cells, yeast CPY is sorted away from secretory proteins in the late Golgi and transits to the vacuole via a distinct endosome-like intermediate.     

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.     

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.     

Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide

We have isolated cis-acting mutations in the gene encoding the yeast vacuolar protein carboxypeptidase Y (CPY) that result in missorting and aberrant secretion of up to 95% of newly synthesized CPY. The CPY polypeptides synthesized by these mutants use the late secretory pathway to exit the cell, since the late-acting sec1 mutation prevents their secretion. The mutant versions of CPY are secreted as the proCPY zymogen and are enzymatically activatable in vivo and in vitro. All the mutations, including small deletions and an amino acid substitution, map to the amino-terminal propeptide region and define a discrete yeast vacuolar localization domain whose integrity is required for efficient sorting of the CPY zymogen. Thus, the N-terminal propeptide of CPY carries out at least three functions: it mediates translocation across the endoplasmic reticulum, renders the enzyme inactive during transit, and targets the molecule to the vacuole.     

Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast

Clathrin heavy chain-deficient mutants (chcl) of Saccharomyces cerevisiae are viable but exhibit compromised growth rates. To investigate the role of clathrin in intercompartmental protein transport, two pathways have been monitored in chcl cells: transport of newly synthesized vacuolar proteins to the vacuole and receptor-mediated uptake of mating pheromone. Newly synthesized precursors of the vacuolar protease carboxypeptidase Y (CPY) were converted to mature CPY with similar kinetics in mutant and wild-type cells. chcl cells did not aberrantly secrete CPY and immunolocalization techniques revealed most of the CPY in chcl cells within morphologically identifiable vacuolar structures. Receptor-mediated internalization of the mating pheromone alpha-factor occurred in chcl cells at 36-50% wild-type levels. The mutant cells were fully competent to respond to pheromone-induced cell-cycle arrest. These results argue that in yeast, clathrin may not play an essential role either in vacuolar protein sorting and delivery or in receptor-mediated endocytosis of alpha-factor. Alternative mechanisms ordinarily may execute these pathways, or be activated in clathrin-deficient cells.     

Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.     

Synthetic genetic interactions with temperature-sensitive clathrin in Saccharomyces cerevisiae. Roles for synaptojanin-like Inp53p and dynamin-related Vps1p in clathrin-dependent protein sorting at the trans-Golgi network

Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone alpha-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in alpha-factor maturation defects and exacerbated alpha-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.     

Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast

Yeast produce two classes of secretory vesicles (SVs) that differ in both density and cargo protein content. In late-acting secretory mutants (e.g. snc1(ala43) and sec6-4), both low- (LDSV) and high-density (HDSV) classes of vesicles accumulate at restrictive temperatures. Here, we have found that disruptions in the genes encoding a dynamin-related protein (VPS1) or clathrin heavy chain (CHC1) abolish HDSV production, yielding LDSVs that contain all secreted cargos. Interestingly, disruption of the PEP12 gene, which encodes the t-SNARE that mediates all Golgi to pre-vacuolar compartment (PVC) transport, also abolishes HDSV production. In contrast, deletions in genes that selectively confer vacuolar hydrolase sorting to the PVC or protein transport to the vacuole (i.e. VPS34 and VAM3, respectively) have no effect. Thus, one branch of the secretory pathway in yeast involves an intermediate sorting compartment and has a specific requirement for clathrin and a dynamin-related protein in SV biogenesis.     

vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting.

We have isolated four yeast mutants that are unable to partition maternal vacuoles into growing buds. Three of these vacuole segregation (vac) mutants also mislocalize the vacuolar protease carboxypeptidase Y (CPY) to the cell surface, a phenotype previously reported for vac strains. A fourth mutant, vac2-1, exhibits a temperature-sensitive defect in vacuole segregation but does not show a defect in protein targeting from the Golgi apparatus to the vacuole. Haploid vac2-1 cells grown at the non-permissive temperature do not secrete CPY or a second vacuolar protease, proteinase A (PrA). Furthermore, newly synthesized precursors of CPY are converted to mature forms with similar kinetics in both vac2-1 and wild-type cells. In addition, invertase is secreted normally from vac2-1 cells, indicating that post-Golgi steps in the secretory pathway are not blocked in this mutant. These results suggest that VAC2 function is necessary for vacuole division and segregation in yeast but is not involved in vacuole protein sorting events at the Golgi apparatus.     

Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast

The mechanism of cargo sorting at the trans-Golgi network (TGN) for secretion is poorly understood. We previously reported the involvement of the actin-severing protein cofilin and the Ca(2+) ATPase secretory pathway calcium ATPase 1 (SPCA1) in the sorting of soluble secretory cargo at the TGN in mammalian cells. Now we report that cofilin in yeast is required for export of selective secretory cargo at the late Golgi membranes. In cofilin mutant (cof1-8) cells, the cell wall protein Bgl2 was secreted at a reduced rate and retained in a late Golgi compartment, whereas the plasma membrane H(+) ATPase Pma1, which is transported in the same class of carriers, reached the cell surface. In addition, sorting of carboxypeptidase Y (CPY) to the vacuole was delayed, and CPY was secreted from cof1-8 cells. Loss of the yeast orthologue of SPCA1 (Pmr1) exhibited similar sorting defects and displayed synthetic sickness with cof1-8. In addition, overexpression of PMR1 restored Bgl2 secretion in cof1-8 cells. These findings highlight the conserved role of cofilin and SPCA1/Pmr1 in sorting of the soluble secretory proteins at the TGN/late Golgi membranes in eukaryotes.     

Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast

The role of clathrin in endocytosis of the yeast phermone receptors was examined using strains expressing a temperature-sensitive clathrin heavy chain. The yeast phermone receptors belong to the family of seven transmembrane segment, G-protein-coupled receptors. A rapid and reversible defect in uptake of radiolabeled alpha-factor pheromone occurred when the cells were transferred to the nonpermissive temperature. Constitutive, pheromone-independent internalization of newly synthesized a-factor phermone receptor was also rapidly inhibited in mutant strains at the nonpermissive temperature. In both cases residual endocytosis, 30-50% of wild-type levels, was detected in the absence of functional clathrin heavy chain. Once internalized, the a- factor receptor was delivered to the vacuole at comparable rates in chc1-ts and wild-type cells at the nonpermissive temperature. Clathrin heavy chain was also required for maximal uptake of a mutant a-factor receptor which is dependent on pheromone for internalization. In the presence of a-factor, the internalization rate of the mutant receptor in chc1-ts cells at the nonpermissive temperature was 2.5 times slower than the rate observed for endocytosis of the mutant receptor in wild- type cells. These experiments provide in vivo evidence that clathrin plays an important role in the endocytosis of the seven trans-membrane segment pheromone receptors in yeast.     

Genetic analysis of clathrin function in yeast

The use of yeast mutants to study the function and dynamics of clathrin-coated membranes has offered new insights into clathrin's role in the secretory pathway and has raised additional questions. Most strains of yeast can incur a disruption of clathrin heavy or light chain genes and remain viable. However, in rare cases, alleles of genes other than clathrin affect the viability of clathrin-deficient cells. The relationship of the products of these genes to clathrin awaits clarification. Phenotypic characterization of clathrin-deficient yeast mutants suggests that clathrin is not essential for the generation of secretory pathway transport vesicles at the ER or the Golgi complex but is required for the intracellular retention of a Golgi membrane protein, Kex2p. With this genetic evidence for clathrin's function in vivo, biochemical and genetic experiments can be designed to address the mechanism by which clathrin effects retention of Kex2p. Clathrin-deficient yeast carry out protein secretion, receptor-mediated endocytosis of mating pheromone, and efficient targeting of newly synthesized vacuolar proteins. These observations challenge aspects of clathrin's proposed involvement in protein transport through the secretory pathway and to lysosomes in mammalian cells. However, the differences are beginning to recede in the face of additional experiments; the formation of clathrin coated vesicles is no longer commonly thought to be obligately coupled to transport through the secretory pathway in mammalian cells (Rothman 1986; Brodsky, 1988), and the role of clathrin in retaining a Golgi membrane protein in yeast may have its precedents in receptor-mediated endocytosis by mammalian cells or in secretory granule formation in endocrine cells. A unified theory of clathrin function is emerging (Brodsky, 1988) which suggests that the clathrin coat assemblage (clathrin heavy and light chains and the associated proteins) acts as a facilitator of intracellular protein transport by sorting and concentrating cargo molecules. The results from studies of clathrin-deficient yeast support this theory. Future experiments will determine whether clathrin provides its functions at different transport stages in different organisms or whether all eukaryotic cells employ clathrin at the same stages of intracellular protein transport.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.