Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p
1997; Springer Nature; Volume: 16; Issue: 8 Linguagem: Inglês
10.1093/emboj/16.8.1820
ISSN1460-2075
Autores Tópico(s)Lysosomal Storage Disorders Research
ResumoArticle15 April 1997free access Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p Markus Babst Markus Babst Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Trey K. Sato Trey K. Sato Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Lois M. Banta Lois M. Banta Haverford College, Department of Biology, Haverford, PA, 19041-1392 USA Search for more papers by this author Scott D. Emr Corresponding Author Scott D. Emr Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Markus Babst Markus Babst Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Trey K. Sato Trey K. Sato Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Lois M. Banta Lois M. Banta Haverford College, Department of Biology, Haverford, PA, 19041-1392 USA Search for more papers by this author Scott D. Emr Corresponding Author Scott D. Emr Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA Search for more papers by this author Author Information Markus Babst1, Trey K. Sato1, Lois M. Banta2 and Scott D. Emr 1 1Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA, 92093-0668 USA 2Haverford College, Department of Biology, Haverford, PA, 19041-1392 USA The EMBO Journal (1997)16:1820-1831https://doi.org/10.1093/emboj/16.8.1820 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4Δ1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome. Introduction The structure and function of the subcellular organelles in eukaryotic cells depend on the specific localization and retention of resident proteins. These proteins are transported from their common site of synthesis to the unique compartment in which they function. A well-studied transport pathway in the yeast Saccharomyces cerevisiae is responsible for delivery of proteins to the vacuole, the functional equivalent of the lysosome in mammalian cells. The biosynthetic transport pathway for the vacuolar enzyme carboxypeptidase Y (CPY) is well characterized and therefore serves as a general model for vesicle-mediated vacuolar protein sorting (reviewed in Stack et al., 1995). The CPY protein is translocated into the endoplasmic reticulum (ER) and subsequently traverses the Golgi complex. In the trans-Golgi, CPY is sorted away from secretory traffic by binding to the receptor protein Vps10p which carries CPY to a pre-vacuolar endosome-like compartment (Marcusson et al., 1994). From this compartment, Vps10p recycles back to the trans-Golgi for further rounds of sorting, while CPY is transported on to the vacuole (Cereghino et al., 1995; Cooper and Stevens, 1996). A similar pathway is found in higher eukaryotes, where lysosomal proteins carrying a mannose-6-phosphate moiety are recognized in the trans-Golgi network by one of the two mannose-6-phosphate receptors and subsequently transported to the endosome (reviewed in Kornfeld, 1992). Multiple transport pathways depend on the endosomal membrane system. Endocytosed proteins are first delivered to an early endosome, where they are either recycled back to the plasma membrane or delivered to the lysosome/vacuole via a late/matured endosome. Newly synthesized vacuolar hydrolases also transit through an endosomal intermediate en route from the trans-Golgi to the lysosome/vacuole. The biosynthetic pathway for lysosomal/vacuolar hydrolases thus converges at the endosome with endocytic traffic (Vida et al., 1993; Schimmöller and Riezman, 1993; for a review, see Gruenberg and Maxfield, 1995). Although the general features of the endosomal pathways are well established, the mechanisms which are responsible for organization and function of the compartment are poorly understood. In S.cerevisiae, several selection schemes have been undertaken to identify mutants defective in the delivery of proteins to the vacuole. This has resulted in the identification of >40 complementation groups of vacuolar protein sorting (vps) mutants (Jones, 1977; Robinson et al., 1988; Rothman et al., 1989). The vps mutants are divided into the classes A–F, based on their vacoular morphology and sorting defects (Banta et al., 1988; Raymond et al., 1992). The 13 class E vps mutants accumulate vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar structure, the class E compartment (Raymond et al., 1992; Davis et al., 1993; Cereghino et al., 1995; Vida and Emr, 1995; Rieder et al., 1996). Previous analysis of two class E mutants, vps27 and vps28, indicated that the class E compartment might represent an exaggerated pre-vacuolar endosomal compartment (Piper et al., 1995; Rieder et al., 1996). We describe here the cloning and characterization of VPS4, a class E VPS gene which encodes a protein that belongs to the family of AAA-type ATPases. The ATPase activity was found to be required for Vps4p function in vacuolar protein trafficking. The phenotypic characterization of a temperature-sensitive vps4 allele indicated that the immediate consequence of loss of Vps4p function is a block in protein and membrane transport from the pre-vacuolar endosome to the vacuole. Results Cloning and sequence analysis of VPS4 The wild-type VPS4 gene was cloned from a yeast genomic library by complementation of the CPY sorting defect of SEY4-17 (method as described in Burd et al., 1996). The complementing activity was localized to a minimal 1.8 kb HindIII fragment. DNA sequencing of this fragment revealed that the complementing fragment contains an open reading frame (ORF) of 1314 bp predicted to encode a protein with a mol. wt of 48 168 (accession No. U25842). The haploid yeast strain MBY4 carrying a deletion in the VPS4 ORF was constructed (vps4Δ1; Materials and methods). The phenotype of this deletion strain and the diploid which resulted from crossing MBY4 with SEY4-17 was indistinguishable from that of SEY4-17, indicating that the cloned fragment indeed corresponds to the VPS4 chromosomal locus. Sequence comparisons with nucleotide databases identified VPS4 homologs in Schizosaccharomyces pombe, mouse and human (Figure 1). Additionally, a 220 amino acid region of the Vps4 protein (Vps4p) was found to be highly homologous to the catalytic domain of AAA-type ATPases. Within this group, the proteins with the strongest overall similarity to Vps4p were Pas8p, a yeast protein involved in peroxisome biogenesis (McCollum et al., 1993), and Yta6p, a possible subunit of the yeast proteosome (Schnall et al., 1994). From the primary amino acid sequence analysis, we postulate a three domain structure for the Vps4 protein: the N-terminal domain, containing a putative ∼30 amino acid coiled-coil motif, the central ATPase domain and the highly charged C-terminal domain (Figure 1). Figure 1.Amino acid sequence alignment of Vps4p homologs from S.cerevisiae (S.c.), Schizosaccharomyces pombe (S.p.; DDBJ/EMBL/GenBank l33456), Mus musculus (M.m.; SKD1, Périer et al., 1994) and Homo sapiens (H.s.; DDBJ/EMBL/GenBank H14152, C03377, M85872, F07485). The sequence alignment was done with the assistance of CLUSTAL W (Thompson et al., 1994). Identities between sequences are indicated with black boxes. The solid line marks the AAA domain, the dots indicate the a and d position of a putative coiled-coil motif predicted by COILS (Lupas et al., 1991). Amino acid changes in the mutant proteins Vps4pts, Vps4pK179A and Vps4pE233Q are indicated beneath the alignment. Download figure Download PowerPoint Vps4p functions in vacuolar protein sorting We analyzed the effect of the VPS4 deletion (vps4Δ1) on the sorting of vacuolar proteins by following the maturation of the vacuolar protein CPY in pulse–chase experiments. Cells were metabolically labeled for 10 min with Trans35S-label and chased for 30 min by the addition of unlabeled cysteine and methionine. The cells were spheroplasted and separated into intracellular and extracellular fractions. CPY was then immunoprecipitated, and the distribution and size of the protein was determined by SDS–PAGE (Rieder et al., 1996). In wild-type cells, the ER-modified form of CPY (p1CPY) is glycosylated further in the Golgi complex, resulting in the p2-form of CPY, and ultimately is transported to the vacuole, where p2CPY is proteolytically cleaved to the mature protein (mCPY). In contrast to wild-type cells, which completely matured CPY during the 30 min chase, the vps4Δ1 strain secreted ∼50% of p2CPY (Figure 2, lanes 7 and 8). To examine morphological changes in the vps4 mutant, cells were stained with the fluorescent dye FM4-64, a lipophilic dye which is internalized and delivered to the vacuole in an energy- and temperature-dependent manner and thus serves as a marker for bulk membrane endocytosis in yeast (Vida and Emr, 1995). Compared with wild-type, vps4Δ1 cells showed fewer but enlarged vacuoles and the appearance of a small, brightly stained compartment adjacent to the vacuole (data not shown). This new structure is probably the class E compartment which accumulates proteins destined for the vacuole as previously characterized for class E vps mutants (Vida and Emr, 1995; Rieder et al., 1996). This result was confirmed by indirect immunofluorescence of the 60 kDa V-ATPase subunit, which was found, consistent with other class E mutants (vps28, Rieder et al., 1996; vps27, Piper et al., 1995), in both the class E compartment and the vacuole of vps4Δ1 cells (data not shown). In summary, the CPY sorting defect and the morphological phenotype of vps4Δ1 mutants are very similar to those described for the original vps4 mutants (Banta et al., 1988; Robinson et al., 1988; Raymond et al., 1992). Figure 2.Sorting of the vacuolar hydrolase CPY analyzed by pulse–chase experiments. Yeast cultures grown at 30°C were labeled with Tran35S-Label for 10 min and harvested 30 min after adding chase. Cells were spheroplasted and separated into intracellular (I) and extracellular (E) fractions. CPY was immunoprecipitated and analyzed by SDS–PAGE. Sorting analysis was performed on wild-type cells SEY6210 (WT) carrying pMB10 (2 μ VPS4) or pMB24 (vps4K179A) and on vps4Δ1 strain MBY3 carrying pMB4 (VPS4) or pMB24 (vps4K179A). Download figure Download PowerPoint Yeast cells harboring VPS4 on a high-copy number (2 μ) plasmid overexpressed Vps4p at least 10-fold (Figure 3, lane 6). This resulted in a dominant-negative phenotype. Approximately 30% of p2CPY was secreted by these cells (Figure 2, lanes 3 and 4), and staining with FM4-64 dye indicated the presence of a class E compartment (data not shown). Thus the overexpression of VPS4 resulted in phenotypes similar to the vps4Δ1 strain. Elevated levels of Vps4p are likely to result in the titration of another protein or proteins required for vacuolar protein sorting. Figure 3.Immunoblot analysis of yeast extracts using anti-Vps4p antibodies. The left panel shows the immunoblot of cell extracts of SEY6210 (WT), MBY3 (vps4Δ1), VPS4 deletion strain MBY3 carrying pMB4 (VPS4) or pMB24 (vps4K179A) or pMB59 (vps4ts) and SEY6210 carrying pMB10 (2 μ VPS4). The right panel shows the fractionation of Vps4p using the wild-type (WT) strain SEY6210 (S100, P100; supernatant and pellet after centrifugation at 100 000 g). Download figure Download PowerPoint Sorting phenotype of a temperature-sensitive VPS4 allele To study the immediate consequences of loss of Vps4p function, we constructed a temperature-sensitive (ts) VPS4 allele. The VPS4 gene was mutagenized using an error-prone PCR mutagenesis technique (see Materials and methods). The mutagenized DNA was cloned into a centromeric plasmid and transformed into the vps4Δ1 strain MBY2. A total of 20 000 colonies were screened using a chromosomally integrated CPY–invertase fusion gene that allows in situ identification of mutants that secrete CPY–invertase at high temperatures (Paravicini et al., 1992). Several vps4ts alleles were generated, and one of them (vps4ts229) was selected for further analysis. By exchanging regions of the wild-type gene with the corresponding vps4ts gene fragment, we mapped the ts mutation(s) to a 309 bp NcoI–NarI fragment that codes for the C-terminal part of the ATPase domain. The sequence of this fragment revealed two adenosine to guanosine substitutions resulting in the exchange of two conserved amino acids (Met307→Thr and Leu327→Ser; Figure 1). The vps4ts plasmid expressed stable Vps4p at levels similar to wild-type VPS4 (Figure 3, lane 5). To assay the vacuolar protein sorting competence of the vps4ts mutant, a vps4Δ1 strain carrying vps4ts on a centromeric plasmid (pMB59) was pre-incubated at either 26 or 37°C for 15 min, then subjected to pulse–chase analysis. The distribution and size of the vacuolar proteins CPY, proteinase A (PrA), alkaline phosphatase (ALP) and carboxypeptidase yscS (CPS) were determined by immunoprecipitation and SDS–PAGE (Figure 4). At 26°C, each vacuolar protein tested was matured normally and remained inside the cell, indicating that at low temperature, vps4ts cells sort these proteins in a manner indistinguishable from wild-type cells (Figure 4, lanes 2, 3, 6 and 7). In contrast, at the non-permissive temperature (pre-shift of 15 min), newly synthesized CPY accumulated as the Golgi-modified p2CPY precursor, and ∼50% was secreted over the course of the chase. The strong block in CPY maturation suggested that the Vps4ts protein was completely inactivated at 37°C (Figure 4, lanes 4 and 5). The intracellular pool of p2CPY migrated at a slightly higher molecular mass than observed in wild-type cells, which might result from hyperglycosylation of CPY. PrA, the other soluble vacuolar hydrolase tested, also accumulated inside the cell as a slower migrating, Golgi-modified form; however, no PrA was secreted in the mutant at high temperature. Analysis of the integral membrane proteins ALP and CPS revealed that in vps4ts cells at high temperature, maturation of CPS was blocked whereas maturation of ALP was unaffected (Figure 4). Figure 4.Vacuolar protein sorting in vps4ts cells. Strain MBY3 pMB4 (WT) and MBY3 pMB59 (vps4ts) were grown at 26°C and used either directly for pulse–chase experiments at 26°C or pre-incubated for 15 min at 37°C prior to pulse–chase analysis (see Figure 2). Before electrophoresis, CPS samples were treated with endoglycosidase H. An intracellular fraction of MBY3 pMB4 (26°C) analyzed either directly after pulse (ALP) or 5 min after addition of chase (CPY, PrA, CPS) served as standards (St) for the different forms (p2, pro, m) of the vacuolar proteins. Download figure Download PowerPoint To study the kinetics of onset of the vps4ts phenotype, we shifted vps4ts cells from permissive to non-permissive temperature for different times prior to pulse–chase ('pre-shift') and followed the sorting of CPY (Figure 5). When vps4ts cells were shifted to 37°C concomitant with the addition of label, CPY maturation was blocked, and p2CPY accumulated inside the cell (lanes 1 and 2). This rapid block in maturation suggests that Vps4p is directly involved in the transport of p2CPY to the vacuole and that the Vps4pts protein loses its function rapidly after shift to the non-permissive temperature. Significant secretion of p2CPY was observed when vps4ts cells were shifted to 37°C 5 min before addition of label, and ∼50% of p2CPY was secreted from cells after pre-shifts longer than 10 min. This sorting defect is identical to that observed with the VPS4 deletion strain (Figure 5, lanes 5 and 6). The delay in onset of CPY secretion indicates that this phenotype is a secondary effect of the loss of Vps4p function. In earlier publications, it was speculated that the reason for p2CPY secretion in class E vps mutants (includes vps4) might be the depletion of the CPY sorting receptor, Vps10p, from the late-Golgi, caused by inefficient recycling of Vps10p from an endosomal compartment back to the Golgi complex (Cereghino et al., 1995; Piper et al., 1995). A prediction of this hypothesis is that overexpression of VPS10 might increase the concentration of Vps10p in the late-Golgi and thus result in reduced secretion of p2CPY. To test this, we introduced VPS10 on a multi-copy plasmid into the vps4ts strain and followed the onset kinetics of p2CPY secretion after shift to the non-permissive temperature (Figure 5, lanes 7–12). As expected, increased expression of VPS10 suppressed the secretion phenotype of the vps4ts cells but did not suppress the block in p2CPY maturation. Figure 5.Kinetic onset of the sorting defect in vps4ts and in a vps4ts strain overexpressing the sorting receptor Vps10p. Cultures of MBY3 pMB59 (vps4ts) or MBY3 pMB59 pEMY10-2 (vps4ts, 2 μ VPS10) were grown at 26°C and shifted for either 0, 5 or 10 min to 37°C (Preshift) prior to pulse labeling for 5 min and chasing for 30 min at 37°C (see Figure 2). Download figure Download PowerPoint vps4 mutants accumulate precursor CPY in a pre–vacuolar compartment To address where CPY transport is blocked in the vps4ts mutant, we monitored by indirect immunofluorescence microscopy the distribution of CPY in vps4ts cells before and after shift to the non-permissive temperature. As expected, the strongest CPY staining at the permissive temperature was localized within the vacuole (Figure 6A and B). However, the vacuolar staining was relatively weak, which might be due to a loss of soluble vacuolar proteins during cell preparation. After 30 min incubation at the non-permissive temperature, the majority of CPY in vps4ts cells was localized in one or two compartments adjacent to the vacuole (Figure 6C and D). These compartments do not resemble the punctate distribution of the Golgi-resident protein Mnn1p (data not shown), and probably represent class E compartments. Figure 6.Cellular distribution of CPY in vps4ts cells analyzed by indirect immunofluorescence microscopy. MBY3 pMB59 cells (vps4ts) were grown at 26°C to an optical density (600 nm) of 0.6 and incubated for 30 min either at 26°C (A and B) or 37°C (C and D) prior to cell preparation (see Materials and methods). Stained cells were visualized by fluorescence (A and C) or Nomarski (B and D) microscopy. Download figure Download PowerPoint To substantiate further the post-Golgi block conferred by vps4ts, we monitored in a pulse–chase experiment intracellular and extracellular CPY at late time points after adding chase. This experiment revealed that the amount of CPY inside the cell was constant over 2 h and that during this time, no additional p2CPY was secreted (Figure 7). Studies of mutants in the CPY sorting receptor Vps10p have shown that a block in transport out of the late-Golgi results in the rapid secretion of p2CPY (Marcusson et al., 1994). Our studies thus suggest that in vps4ts cells at the non-permissive temperature, CPY does not accumulate in the late-Golgi but instead is sorted to a more distal compartment like the pre-vacuolar endosome that accumulates in class E vps mutants. Figure 7.Maturation of CPY in vps4ts cells. MBY3 pMB59 cells (vps4ts) were grown at 26°C and shifted to 37°C 2 min prior to a 10 min pulse labeling. Samples were harvested at the times indicated after addition of chase and further incubated at 37°C. The analysis was performed as described in Figure 2. Different forms of CPY are marked (p2, m, *). Download figure Download PowerPoint Unlike the vps4Δ1 strain, which accumulated mCPY inside the cell, vps4ts cells accumulated exclusively p2CPY at early time points after shift to the non-permissive temperature (compare Figure 2, lane 7 with Figure 4, lane 4). This difference could be explained by the fact that in vps4Δ1 cells, p2CPY is transported from the Golgi to a class E compartment, which contains active proteases and is competent to mature p2CPY (Raymond et al., 1992; Piper et al., 1995). In contrast, at early time points, vps4ts cells might not have yet formed a proteolytically active class E compartment, hence no p2CPY is matured. To test this, we followed maturation of CPY at late time points after adding chase (Figure 7). The p2CPY which accumulated inside the cell after shift to the non-permissive temperature was processed within 2 h to the mature form. Maturation occurred via an intermediate form which was not observed in wild-type cells (see *, lane 5 in Figure 7). This suggests that the maturation step did not occur in the vacuole but instead was a consequence of formation of a proteolytically competent class E compartment. In vps4ts cells which were pre-incubated at the non-permissive temperature for 2 h prior to the pulse–chase experiments, 70% of the newly synthesized, intracellular CPY matured during the 30 min chase (data not shown). This result indicates that after 2 h at non-permissive temperature, newly synthesized CPY was transported to a proteolytically competent class E compartment. The transport defect in vps4ts cells is reversible To test if the block in CPY transport observed in vps4ts cells is reversible, we shifted the cells to 37°C, labeled them for 5 min, and then chased them for 15 min at 37°C. The culture was then shifted back to the permissive temperature, and samples were taken at various time points (Figure 8A). CPY and Vps4p were immunoprecipitated and analyzed by SDS–PAGE (Figure 8B). Most of the p2CPY which accumulated in a pre-vacuolar compartment during incubation at the non-permissive temperature was processed to the mature form within 30 min after shift to the permissive temperature. Compared with the processing observed after a long chase at the non-permissive temperature (Figure 7), CPY maturation observed in this experiment occurred faster and no processing intermediate was observed. This indicated that CPY was transported to the vacuole. The amount of Vps4pts was constant during the experiment (Figure 8), suggesting that the temperature sensitivity of Vps4pts was not caused by rapid degradation of the protein at high temperature. Furthermore, we found that the addition of cycloheximide to the culture (100 mg/l) prior to the shift back to 26°C did not influence the maturation kinetics of CPY (data not shown), which argues that de novo Vps4p synthesis is not necessary for the observed reversal of the vps4ts defect. In summary, these data demonstrate that both the loss of Vps4pts function and the block in CPY transport are reversible. Figure 8.Reversibility of the sorting phenotype of vps4ts cells. Cultures of the strains MBY3 pMB59 (vps4ts) and MBY3 pMB4 (WT) were grown at 26°C and shifted to 37°C; at the same time label was added (P). After 5 min, chase was initiated (C), and the cells were incubated further at 37°C for 15 min. The culture was then shifted back to 26°C, and samples were harvested at the time points indicated (S1–S4; A). CPY from the extracellular (E) and CPY and Vps4p from the intracellular (I) fractions were analyzed by immunoprecipitation and SDS–PAGE (B). Download figure Download PowerPoint Morphology of vps4ts cells Previous characterization of class E vps mutants revealed the presence of stacks of curved cisternal membranes (Rieder et al., 1996) known as the class E compartment that is thought to represent an exaggerated pre-vacuolar intermediate compartment (Piper et al., 1995; Rieder et al., 1996). The vps4ts mutants allowed us to observe the formation of the class E compartment. We characterized by electron microscopy the morphology of vps4ts cells which were grown at 26°C and shifted to 37°C for various lengths of time. Analysis of >150 sections of cells grown at the permissive temperature revealed the appearance of only a few (<5%) membrane structures similar to class E compartments (Rieder et al., 1996). Several sections of these cells contained aberrant membrane stacks, most of which were adjacent to the vacuole (see arrow in Figure 9A). After 1 h at the non-permissive temperature, the number of cell sections containing stacks of 4–6 curved cisternal membranes increased from <5% to ∼30% (n = 150; Figure 9B–E). The morphology and frequency of these class E structures is comparable with observations reported in a previous study of a VPS28 deletion mutant (Rieder et al., 1996). Figure 9.Electron microscopic analysis of vps4ts cells at the permissive and non-permissive temperature. (A) Cross-section of an MBY3 pMB59 cell (vps4ts) grown at 26°C. The arrow marks aberrant membrane stacks that accumulate in this strain. (B) A cell from the same culture after incubation for 1 h at 37°C. (C–E) Examples of class E compartments formed during 1 h incubation at the non-permissive temperature. (E) The enlarged class E compartment seen in (B). The scale bars in (A) and (B) and (C–E) represent 0.5 and 0.2 μm, respectively (m, mitochondria; v, vacuole). Download figure Download PowerPoint Vps4p is involved in the endocytic pathway Several studies have demonstrated that class E mutants exhibit a block in the endocytic pathway (vps2, Davis et al., 1993; vps4, Munn and Riezman, 1994; vps27, Piper et al., 1995; vps28, Rieder et al., 1996), which might be a secondary defect due to the accumulation of aberrant endosomal structures. We tested if loss of Vps4p activity directly affects endocytic traffic by comparing Ste6p stability in vps4ts and wild-type cells. Ste6p, the transporter of the mating pheromone a-factor, is rapidly removed from the plasma membrane by endocytosis and subsequently is degraded in the vacuole (Kölling and Hollenberg, 1994). Cells were grown at 26°C and shifted to 37°C 10 min prior to the pulse–chase experiment. The Ste6 protein was immunoprecipitated after different chase times and analyzed by SDS–PAGE (Figure 10). This experiment showed a 2- to 3-fold stabilization of Ste6p in vps4ts compared with wild-type cells. This suggests that in vps4ts cells, the transport of Ste6p to the vacuole, where degradation occurs, was impaired. Earlier studies indicated the existence of more than one pathway for the degradation of Ste6p which would explain why in vps4ts cells Ste6p was only partially stabilized (Kölling and Hollenberg, 1994). Figure 10.Stability of Ste6p in wild-type and vps4ts. Cultures of MBY4 pMB4 pDB192 (WT) and MBY4 pMB59 pDB192 (vps4ts) were grown at 26°C, shifted to 37°C 10 min prior to the pulse labeling for 15 min. After addition of chase (time 0), the culture was incubated further at 37°C, and samples were harvested at the time points indicated (Chase). The Ste6 protein from these samples was immunoprecipitated and analyzed by SDS–PAGE. Download figure Download PowerPoint The ATPase activity of Vps4p is critical for sorting of vacuolar proteins To determine whether the AAA motif of Vps4p is essential for proper vacuolar protein sorting, we used site-directed mutagenesis to change the codons of a highly conserved lysine (position 179) and glutamate (position 233) in the ATPase domain to a codon for alanine or glutamine, respectively (Vps4pK179A, Vps4pE233Q; Figure 1). Analogous amino acid changes have been shown previously to abolish ATPase activity and function of the NEM-sensitive fusion protein (NSF) (Whiteheart et al., 1994). Western blot analysis showed that Vps4pK1
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