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Vol. 16, Issue 4, 1673-1683, April 2005
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* Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755;
Department of Molecular Medicine, Cornell University, Veterinary Medical Center, Ithaca, NY 14853
Submitted October 7, 2004;
Accepted January 10, 2005
Monitoring Editor: Benjamin Glick
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). In this way, secretory cargos are directed to their correct cellular destination, whereas donor and acceptor organelles maintain their specific protein and lipid compositions. Specific sets of cytosolic coat proteins mediate the biogenesis of transport carriers, whereas conserved membrane fusion factors such as Rabs and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) are believed to mediate site-specific fusion of transport vesicles with acceptor compartments.
Although many of the protein factors required for intracellular transport have been identified and characterized, it is less clear how these factors interact to coordinate the maintenance of the secretory pathway. In the budding yeast Saccharomyces cerevisiae, transport between the endoplasmic reticulum (ER) and the Golgi complex also relies on coat proteins, Rabs, and SNAREs. Specifically, the Rab protein Ypt1p is thought to play a key role in the tethering of ER-derived transport vesicles to the Golgi apparatus (Segev, 1991; Cao et al., 1998). Yeast two-hybrid approaches identified a Ypt1p-interacting protein (Yip1p) required for transport through the early secretory pathway (Yang et al., 1998). Yip1p is an essential protein of 27-kDa and is predicted to contain three transmembrane segments. Yip1p is conserved across species, and a family of Yip1p-like proteins has recently been described in humans (Tang et al., 2001; Shakoori et al., 2003). The NH2 terminus of Yip1p projects into the cytosol and is proposed to be sufficient for interaction with Ypt1p (Yang et al., 1998). Yip1p localizes to Golgi and ER membranes as well as ER-derived COPII transport vesicles, indicating Yip1p cycles between these compartments (Heidtman et al., 2003).
Additional studies showed that Yip1p forms a heteromeric complex with a second multispanning integral membrane protein termed Yif1p (Matern et al., 2000). Yif1p shares sequence homology with Yip1p and also possesses a similar topology. Like Yip1p, Yif1p is able to bind Rab GTPases and is required for transport through the early secretory pathway (Matern et al., 2000). Yeast cells also have two nonessential proteins with homology to Yip1p and Yif1p. These proteins, termed Yip4p and Yip5p, were proposed to constitute additional members of a Yip1p-like family of proteins in yeast and may act at later stages of intracellular transport (Calero et al., 2002). Although much has been learned about Yip1p and related proteins, their precise mechanism of action in the early secretory pathway remains to be elucidated.
We recently investigated the function of Yip1p in transport between the ER and the Golgi complex by using biochemical, genetic, and morphological approaches (Heidtman et al., 2003). Using a cell-free assay driven by purified protein factors that recapitulates protein transport between the ER and the Golgi complex, we found that inhibitors of Yip1p blocked transport at the COPII-dependent budding stage. In addition, a strain bearing the thermosensitive yip1-4 allele accumulated ER membranes but not transport vesicles at the restrictive temperature, corroborating a role for Yip1p in vesicle budding. We concluded that Yip1p plays a critical role in the biogenesis of ER-derived COPII transport vesicles (Heidtman et al., 2003).
To gain further insight into Yip1p function, we have taken a genetic approach. Using a YEp24 yeast genomic library, we selected for multicopy suppressors of the yip1-4 allele. A multicopy suppression screen can be a tool to identify gene products that function in the same process as a gene of interest and can therefore provide important insights into function (Carlson and Botstein, 1982). Here, we describe the identification of the open reading frame YER074W-A as a novel multicopy suppressor of the yip1-4 strain. We show that this gene encodes a conserved, essential integral membrane protein that localizes to Golgi and ER membranes as well as COPII transport vesicles. We have termed this gene Yip One Suppressor 1 (YOS1) and demonstrate that Yos1p physically associates with the Yip1p–Yif1p complex. Cells depleted of Yos1p display secretory defects characteristic of a block in transport between the ER and the Golgi complex, indicating Yos1p is required for this process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and cloning protocols (Ausubel et al., 1987) were used.
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Multicopy Suppressor Screen
For the multicopy suppression screen of the yip1-4 strain, 12,000 transformants of a yeast genomic YEp24 library (Carlson and Botstein, 1982) were grown on YMD-Ura plates at 25°C. Colonies were then replica plated to YPD plates. The replica YPD plates were incubated for 48 h at 34°C. Fifty-five colonies were observed to grow at the restrictive temperature. Cells from each temperature-resistant transformant were restuck to YMD-Ura plates to check that each of the isolates contained plasmid DNA. The putative suppressor strains were then grown overnight in selective medium, and plasmid DNA was isolated. The isolated plasmid DNA was retransformed into the yip1-4 strain to check for plasmid-linked suppression. Of the 55 isolated plasmid preparations, 22 were able to confer temperature resistance to the yip1-4 strain upon retransformation. The ends of the genomic inserts of these 22 suppressing plasmids were then sequenced with primers YEpF1–5'CTAGCGCTATATGCGTTG3'and YEpR3 5'GTGATGTCGGCGA TATAGG3'.
Plasmid Construction
Point mutations in YOS1 were introduced by QuikChange mutagenesis (Stratagene, La Jolla, CA) by using primers containing the desired changes and plasmid pRS314-YOS1 as the template. Mutant plasmids were sequenced using the primer SYMF1 (5'GCTGGACTGGGAGCCT TCTCAATAAG 3'). Mutant plasmids were transformed into strain CBY1781. Transformants were struck to YMD-Trp supplemented with 5-fluororotic acid (5-FOA) (0.1% final concentration) to cure the strain of wild-type (WT) YOS1.
Antibodies and Immunoblotting
Antibodies against Sec61p, Och1p, carboxypeptidase Y (CPY), Sec23p (Otte et al., 2001), Sec22p (Liu and Barlowe, 2002), 
1,6-mannose linkages, Ypt1p (Cao et al., 1998), Yip1p (Heidtman et al., 2003), and Gas1p (Belden and Barlowe, 1996) have been described previously. Polyclonal antibodies were raised against a glutathione S-transferase (GST)-Yos1p (aa 33–66) fusion protein expressed from plasmid pGEX-2T-YOS1 in Escherichia coli. The fusion protein was purified according to the manufacturer's specifications (Pfizer, Inc., New York, NY) and used to immunize rabbits by standard procedures (Covance, Berkeley, CA). Anti-hemagglutinin (HA) monoclonal antibody (mAb) (HA.7) raised against the peptide YPYDVPDYA was obtained from Sigma-Aldrich (St. Louis, MO). For densitometric analysis of immunoblots, films were scanned and plotted using NIH Image 1.52.
Subcellular Fractionation
For experiments to determine the subcellular distribution of Yos1p and Yip1p, organelles were resolved on sucrose gradients according to Powers and Barlowe (1998). To characterize the membrane association of Yos1p, wild-type microsomes were treated with buffer 88 (20 mM HEPES, pH 6.8, 250 mM sorbitol, 150 mM KOAc, and 5 mM MgOAc), 0.1 M sodium carbonate, or 1% Triton X-100 in buffer 88. Samples were mixed and incubated on ice for 10 min, followed by centrifugation at 60,000 rpm (model TLA 100.3 rotor; Beckman Coulter, Fullerton, CA) for 12 min. Equivalent amounts of total samples before centrifugation, supernatant and pellet fractions were diluted in SDS-PAGE buffer, and resolved on a 15% acrylamide gel and analyzed by immunoblot.
Immunoprecipitations
For immunoprecipitation of Yif1p-3xHA or Yos1p, 300 µl of microsomes (320 µg of total membrane protein) was solubilized in an equal volume of 0.5% digitonin/buffer 88-8 (Kuehn et al., 1998) in the presence of 10 mM phenylmethylsulfonyl fluoride and 5 mM EDTA. After centrifugation at 14,000 rpm for 4 min at room temperature (RT) to remove unsolubilized material, the supernatant fluid (
500 µl) was transferred to a fresh tube on ice. The solubilized material was diluted with 1.5 volumes of 0.05% digitonin/buffer 88-8, and HA-tagged proteins were immunoprecipitated by addition of 2.0 µg of anti-HA mAb and 25 µl of 20% protein A-Sepharose beads. For immunoprecipitation of Yos1p, 0.5 µl of anti-Yos1p polyclonal antiserum was used in combination with 25 µl of 20% protein A-Sepharose beads. After binding for 2 h at 4°C, beads with bound protein were washed four times with 0.05% digitonin/buffer 88-8. Finally, the bound protein was released from the beads by the addition of 30 µl of SDS-PAGE sample buffer and heated at 75°C for 3 min. Complexes from the immunoprecipitates were resolved on polyacrylamide gels and analyzed by immunoblot. For immunoprecipitation of Yip1p, 0.5 µl of anti-Yip1p polyclonal antiserum was used in combination with 25 µl of 20% protein A-Sepharose beads in the manner described above.
In Vivo Labeling
Pulse-chase experiments were performed as described previously (Belden and Barlowe, 1996), with minor differences. Wild-type and yos1-1 cells were grown at 25°C in minimal medium containing 2% glucose to an OD600 of 0.4. Cultures were harvested, washed, and resuspended at 1/10 the original volume in minimal medium lacking sulfate. After preculturing for 5 min at the permissive (25°C) and restrictive (37°C) temperatures, cells were pulsed for 7 min by the addition of [35S]methionine (Amersham Biosciences, Piscataway, NJ) and chased by the addition of excess unlabeled methionine. Cell samples were taken at the end of the pulse period and after 30 min of chase. Cell lysates were prepared by bead-beat lysis, and labeled species were precipitated from a common extract with specific antibodies for CPY.
In Vitro Vesicle Budding, Tethering, and Transport Assays
Yeast semi-intact cells from wild-type and mutant strains were prepared as described previously (Baker et al., 1988). Vesicle budding, tethering, and fusion assays following [35S]gpf were published previously (Barlowe, 1997; Cao et al., 1998). Experiments to assay packaging of proteins into vesicles by Western blot were performed as described previously (Otte et al., 2001) from microsomes. For in vitro assays, data points are the average of duplicate determinations and the error bars represent the range.
Microscopy
For analysis of wild-type and yos1–1 cells by electron microscopy (EM), cells were harvested on filters after shift to the restrictive temperature (1 h, 37°C). Cells were then washed once with buffer (0.1 M PIPES, 0.1 M sorbitol, 1 mM CaCl2, and 1 mM MgCl2, pH 6.8), fixed with 1.25% glutaraldehyde in buffer at RT for 1 h, and then overnight at 4°C. Permanganate fixation and staining was performed according to the procedure of Kaiser and Schekman (1990).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To extend these studies and gain further insight into the mechanism of Yip1p action, a genetic approach was undertaken. Specifically, a yeast genomic YEp24 library bearing the URA3 marker (Carlson and Botstein, 1982) was used to select for genes that could rescue the growth defect of the yip1-4 strain at the nonpermissive temperature of 34°C. Approximately 12,000 transformants were selected on minimal plates lacking uracil at the permissive temperature. Transformants were then replica plated to rich media and incubated at 34°C for 48 h. Fifty-five colonies were found to grow at the restrictive temperature. Plasmid DNA was recovered from these colonies and used to retransform the yip1-4 mutant to test for plasmid-linked suppression. Twenty-two of the 55 plasmids were found to confer suppression of the growth defect in the yip1-4 strain at the restrictive temperature upon retransformation. DNA sequencing identified the genomic insert present in each of the suppressing plasmids. Two of these plasmids contained a region of DNA harboring the YIP1 open reading frame (ORF). The remaining 20 plasmids all contained a similar region of genomic DNA from chromosome V. Inspection of this region revealed three ORFs common to all 20 plasmids. These ORFs were ALD5, RPS24, and YER074W-A. Subcloning revealed that YER074W-A was solely responsible for the suppression of the growth defect in the yip1-4 strain at 34°C. We have termed this gene YOS1 based on the manner in which this gene was identified.
To more closely examine the high copy suppression activity of YOS1 with regard to yip1-4, we performed dilution series experiments. Strains were grown overnight in selective medium to stationary phase, back diluted to an equivalent OD600, and a 10-fold dilution series was spotted onto YPD plates. As shown in Figure 1A, the yip1-4 strain transformed with empty vector displayed a severe growth defect after 48 h at 34°C. In contrast, multi-copy YOS1 was able to significantly rescue this growth defect. We next examined whether multi-copy YOS1 was able to rescue any of the secretion defects present in the yip1-4 strain. We previously reported that membranes prepared from the yip1-4 strain display a significant defect in a cell-free assay that measures the COPII-dependent budding of [35S]glyco-pro- factor (Heidtman et al., 2003). Using this assay, we examined the budding efficiencies of washed semi-intact cell membranes prepared from yip1-4 cells bearing empty vector or multicopy YOS1. As shown in Figure 1B, wild-type membranes displayed a budding efficiency of 30%. In contrast, yip1-4 membranes displayed a budding efficiency of only 12%, consistent with our previously published results (Heidtman et al., 2003). Interestingly, we observed that membranes prepared from the yip1-4 strain containing multi-copy YOS1 produced a budding efficiency of 23%. This result indicates that multi-copy YOS1 can partially rescue the budding defect displayed by the yip1-4 membranes in vitro and suggests that the suppression activity of YOS1 with regard to the yip1-4 allele is direct.
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YOS1 is an uncharacterized ORF identified by comparison of the S. cerevisiae genome to that of the related fungi Ashbya gossypii (Blandin et al., 2000). YOS1 contains two introns and is predicted to encode a protein of 87 amino acids with a molecular mass of 9.5 kDa. As shown in Figure 2, Yos1p is well conserved across species, possessing homologues in higher eukaryotic organisms including humans.
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Yos1p Is an Essential Protein Required for Protein Transport between the ER and the Golgi Complex
YOS1 has not been analyzed by previous large-scale studies that sought to classify yeast ORFs as either essential or nonessential for growth. Therefore, we next examined whether YOS1 is essential for growth in yeast cells. A polymerase chain reaction (PCR)-based approach (Longtine et al., 1998) was used to direct the S. pombe his5+ gene to the YOS1 locus, replacing the entire YOS1 ORF. Colony PCR was performed to confirm that the his5+ gene had integrated correctly at the YOS1 locus (our unpublished data). A heterozygous YOS1/yos1
diploid strain was then transformed with empty vector, or a centromere (CEN)-based plasmid containing YOS1. As shown in Figure 3A, dissection of asci from the heterozygous deletion strain containing empty vector resulted in only two viable spores in each case. Tetrad analysis of the surviving spores confirmed that none possessed the his5+ marker, indicating that YOS1 is essential for viability. As shown in the right panel of Figure 3A, inviability could be rescued when the heterozygous deletion strain was transformed with YOS1 on a single-copy plasmid. In this case, three and sometimes four viable spores could be recovered. This observation indicates that the 2+:2– segregation pattern observed in the presence of empty vector is a direct consequence of the deletion of YOS1.
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Because YOS1 is an essential gene, we next created a conditional allele of YOS1. YOS1 was placed under transcriptional control of the GAL1 promoter, allowing for the depletion of Yos1p from cells in glucose-containing medium (Longtine et al., 1998). Cells containing GAL1-YOS1 grew normally in galactose-containing medium but displayed a severe growth defect 6 h after shift to medium containing 2% glucose. Cells depleted of Yos1p, as monitored with a specific anti-Yos1p polyclonal antiserum, accumulated the proforms of secretory proteins en route to various cellular destinations (Figure 3B). Specifically, the core-glycosylated ER form (p1) of the vacuolar hydrolase CPY was observed to accumulate in the GAL1-YOS1 strain 5 h after shift to glucose. Similarly, the core-glycoslyated ER form (p) of the glycosylphosphatidylinositol-anchored plasma membrane protein Gas1p also was observed to accumulate in cells depleted of Yos1p. The ER proform of vacuolar alkaline phosphatase also was seen to accumulate under conditions of Yos1p depletion (our unpublished data). Together, these results indicate that Yos1p is an essential protein in yeast and is required for transport of secretory proteins from the ER to the Golgi complex.
Yos1p Is an Integral Membrane Protein
Hydrophobicity plotting (Kyte and Doolittle, 1982) shows that Yos1p contains two potential transmembrane domains located at the NH2 and COOH termini. These potential transmembrane segments are separated by a relatively hydrophilic stretch of residues (Figure 4A). We sought to confirm that Yos1p is an integral membrane protein by using Western blotting in combination with cellular fractionation under various conditions. As shown in Figure 4B, Yos1p pelleted with membranes when cells were treated with buffer, or with buffer at pH 11, which efficiently extracts the peripherally associated membrane protein Sec23p into the soluble fraction. Yos1p shifted to the soluble fraction when membranes were treated with 1% Triton X-100, in a manner similar to the known integral membrane protein Yip1p. These results confirm that Yos1p is an integral membrane protein. Further experimentation will be required to establish the membrane topology of Yos1p.
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Yos1p Localizes to the Early Secretory Pathway
Because Yos1p was identified as a multicopy suppressor of the yip1-4 strain, we hypothesized that Yos1p would exhibit a similar subcellular localization pattern as Yip1p. Previous studies have shown that Yip1p and Yif1p localize to ER and Golgi membranes as well as COPII transport vesicles (Yang et al., 1998; Matern et al., 2000; Heidtman et al., 2003). In addition, a thermosensitive allele of sec12 that blocks ER export but allows retrograde trafficking causes the accumulation of both Yip1p and Yif1p in ER-enriched membrane fractions (Heidtman et al., 2003). Together, these observations indicate that Yip1p and Yif1p cycle between ER and Golgi membranes. Therefore, we examined the subcellular localization of Yos1p by resolution of membrane organelles on sucrose gradients. As shown in Figure 5A, Yos1p sedimented in one broad peak that overlapped with the Golgi marker Och1p and the ER marker Sec61p. This localization pattern was very similar to that of Yip1p, which also sedimented in one broad peak overlapping with both ER and Golgi markers.
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The observation that Yos1p sediments with both Golgi and ER membranes suggests that Yos1p cycles between these membranes and is therefore packaged into COPII transport vesicles. To test this hypothesis, we prepared microsomes from wild-type cells for use in an in vitro budding reaction. Microsomes were incubated with an energy regeneration system in the presence or absence of COPII proteins, and the vesicles synthesized in each condition were then isolated and analyzed by immunoblotting. As shown in Figure 5B, Yos1p was packaged into vesicles in a COPII-dependent manner, at efficiencies similar to that of Yip1p and the SNARE protein Sec22p. The ER-resident protein Sec61p was not packaged into vesicles in the presence of COPII proteins, demonstrating the selective sorting in this budding assay. We conclude that Yos1p localizes to Golgi and ER membranes and is efficiently packaged into ER-derived COPII transport vesicles.
Yos1p Forms a Complex with Yip1p and Yif1p
Our localization studies indicate that Yos1p displays a similar subcellular distribution to that of Yip1p and Yif1p. In addition, overproduction of Yos1p is able to rescue the growth defect of the yip1-4 strain at the restrictive temperature. These observations suggest that Yos1p function is closely linked to that of Yip1p and therefore Yos1p may physically associate with the Yip1p–Yif1p complex. To test this hypothesis, we prepared microsomes from a strain expressing Yif1p-3xHA as the sole source of Yif1p in the cell. This COOH-terminally tagged version of Yif1p is under transcriptional control of the endogenous YIF1 promoter and fully complements growth (Otte et al., 2001). Microsomes also were prepared from an untagged, isogenic wild-type strain to serve as a negative control. Microsomes from each strain were solubilized at room temperature with 0.5% digitonin. Solubilized extracts were harvested from each strain and subjected to immunoprecipitation with monoclonal anti-HA antibodies in the presence of protein A-Sepharose beads. As shown in Figure 6A, Western blotting revealed that Yif1p-3xHA could be efficiently immunoprecipitated from a solubilized extract generated from the tagged strain. Yip1p was enriched in the immunoprecipitated fraction, confirming the observation that Yip1p and Yif1p form a complex (Matern et al., 2000). Strikingly, Yos1p also was efficiently coimmunoprecipitated under these conditions, indicating that Yos1p was physically associated with the Yip1p–Yif1p complex. The integral membrane protein Sec61p and the SNARE protein Sec22p were not coimmunoprecipated with Yif1p-3xHA, demonstrating that the association of Yos1p with Yip1p–Yif1p was specific. We also analyzed the immunoprecipitates for the presence of Ypt1p but could not detect this protein in these fractions.
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To further explore the observation that Yos1p physically interacts with Yip1p–Yif1p, we performed additional immunoprecipitation experiments. Specifically, a digitonin solubilized extract was generated from the YIF1–3xHA strain and subjected to immunoprecipitation with polyclonal antibodies directed against Yos1p. As shown in Figure 6B, Yos1p was efficiently immunoprecipitated. Both Yip1p and Yif1p-3xHA could be coimmunoprecipitated with Yos1p under these conditions, providing a second line of evidence for the association of Yos1p with Yip1p–Yif1p. Sec61p and Ypt1p were both absent from the immunoprecipitated fractions, confirming the specificity of these interactions. Densitometry analysis of the immunoblot in Figure 6A indicated that 9% of the total Yif1p-3xHA was immunoprecipitated from the soluble extract, with 5% of Yip1p and Yos1p coimmunoprecipitating. This observation suggests a stoichometric association of each subunit of the complex; however, further biochemical analysis of the complex will be required to prove this point. Together, these results demonstrate that Yos1p is physically associated with Yip1p–Yif1p and suggests that Yos1p is a novel subunit of the Yip1p–Yif1p complex. Furthermore, this observation is entirely consistent with the subcellular localization of Yos1p as well as the ability of Yos1p to function as a multicopy suppressor of the yip1-4 strain.
Yos1p Interaction with Yip1p Is Disrupted in the yip1-4 Strain
We next analyzed the nature of complex formation between Yos1p and Yip1p in the yip1-4 strain. We immunoprecipitated Yos1p from either wild-type or yip1-4 digitonin solubilized membranes and monitored the amount of Yip1p and other proteins that coimmunoprecipitated. As seen in Figure 7, Yip1p efficiently coimmunoprecipitated with Yos1p from wild-type–soluble extracts. Interestingly, Yip1p was not co-immunoprecipated with Yos1p from yip1-4 extracts. This observation suggests that complex formation between Yos1p and Yip1p in the yip1-4 strain is disrupted or weakened.
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Mutant yos1-1 Cells Display Transport Defects In Vivo and In Vitro
To further characterize Yos1p function in the early secretory pathway, we undertook a directed mutagenesis approach to generate thermosensitive alleles of YOS1. Conserved residues were mutated to either an alanine or to a residue of opposite property. These mutant versions of Yos1p were then expressed as the sole source of Yos1p by using a plasmid-shuffling approach. The results of the mutagenesis are shown in Table 2. Interestingly, mutation of the invariant asparagine at position 18 to alanine in the first transmembrane domain of Yos1p resulted in a strain that was thermosensitive for growth. We have termed this mutant allele yos1-1. The yos1-1 cells grew near wild-type rates at 30°C but displayed a severe growth defect when shifted to 37°C. We examined the transport kinetics of secretory proteins in this strain compared with wild-type cells. A pulse-chase experiment was performed in which cells were grown in minimal media and pulsed for 7 min with [35S]methionine to label newly synthesized proteins. Excess unlabeled methionine was added for the chase phase, and maturation of vacuolar CPY was monitored by immunoprecipitation with an anti-CPY antibody. CPY first occurs in the ER as the P1 precursor form of 67 kDa and is then modified in the Golgi to produce the P2 form of 69 kDa. Finally, CPY is processed in the vacuole to yield a mature form of 61 kDa (Stevens et al., 1984). As seen in Figure 8A, the yos1-1 strain exhibited a significant delay in the transport of CPY compared with wild-type cells, even at the permissive temperature of 25°C. Strikingly, the yos1-1 strain exhibited a complete block in the transport of CPY at the restrictive temperature of 37°C. This observation further supports the argument that Yos1p is required for protein transport between the ER and the Golgi complex and is consistent with the phenotypes observed by GAL1-regulated depletion of Yos1p from cells.
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We next characterized the yos1-1 strain by using a reconstituted cell-free assay that measures protein transport to the Golgi complex. For this assay, washed semi-intact cell membranes containing [35S]gpf in the ER are incubated with purified factors (COPII, Uso1p, and LMA) to drive transport of [35S]gpf to the Golgi complex (Barlowe, 1997). On delivery to the Golgi complex, gpf receives outer-chain 1,6-mannose residues that can be immunoprecipitated with 1,6-mannose–specific serum to quantify [35S]gpf transport (Baker et al., 1988). As shown in Figure 8B, yos1-1 membranes displayed a modest transport defect of [35S]gpf to the Golgi complex at 25°C. Specifically, the yos1-1 membranes displayed a 30% reduction in [35S]gpf transport compared with wild-type membranes. A similar result was observed when these strains were compared at 29°C; therefore, we were unable to replicate the thermosensitivity of the yos1-1 allele in vitro (our unpublished data).
Subreactions in cell-free transport can be monitored by following the sedimentation properties of membranes containing [35S]gpf (Barlowe, 1997). Incubation of washed semi-intact cell membranes with purified COPII proteins catalyzes the formation of diffusible vesicles that can be separated from larger membranes by centrifugation. When purified Uso1p is included in this reaction, a fraction of the diffusible vesicles now pellet with heavier membranes, allowing a measurement of vesicle tethering. We examined the ability of the yos1-1 strain to bud and tether COPII vesicles to identify the stage at which transport was compromised (Figure 8C). On the addition of COPII proteins, wild-type membranes budded vesicles at an efficiency of 44%, whereas the yos1-1 membranes budded vesicles at an efficiency of only 31%. When the tethering factor Uso1p was included in these reactions, both wild-type and yos1-1 membranes tethered 23% of the diffusible vesicles. These results indicate that the yos1-1 membranes can effectively tether COPII vesicles to the Golgi complex but are compromised in their ability to bud COPII vesicles from ER membranes.
Yip1p Interaction with Yos1p Is Disrupted in the yos1-1 Strain
We next sought to characterize the interaction of Yip1p with Yos1p in the yos1-1 strain. Yip1p was immunoprecipitated from either wild-type or yos1-1 digitonin-solubilized membranes. The amount of Yos1p and other proteins that was coimmunoprecipitated was then monitored by immunoblotting. As shown in Figure 9, Yos1p efficiently coimmunoprecipitated with Yip1p from wild-type soluble extracts. In contrast, Yos1p was not efficiently coimmunoprecipitated with Yip1p from yos1-1 extracts. This result indicates that the interaction between Yip1p and Yos1p in the yos1-1 strain is destabilized or disrupted. This result is consistent with the observation that the interaction between Yip1p and Yos1p also seems to be disrupted in the yip1-4 strain (Figure 7).
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Mutant yos1-1 Cells Display Morphological Phenotypes Characteristic of a Block in the Early Secretory Pathway
We further analyzed the yos1-1 strain by thin-section EM to characterize the morphology of any transport intermediates that may accumulate at the restrictive temperature. As shown in Figure 10, yos1-1 cells displayed morphological phenotypes consistent with a block in the early secretory pathway upon shift to 37°C for 60 min. Specifically, the yos1-1 cells displayed tubulation and/or dislocation of ER membranes from the cell periphery (Figure 10, B and C, white arrows). In addition, clusters of vesicular or tubular elements were sometimes observed (Figure 10D, white arrow). Similar alterations in membrane organization were observed in the yos1-1 strain after a 30-min shift to 37°C. These morphological phenotypes are consistent with a requirement for Yos1p function in the early secretory pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Additional studies showed Yip1p forms a complex with a related integral membrane protein termed Yif1p (Matern et al., 2000). Like Yip1p, Yif1p also displays the ability to bind Rab GTPases in the two-hybrid system. These observations coupled with the localization of Yip1p–Yif1p to the early secretory pathway led to the suggestion that this complex may function as an integral membrane receptor for Rab GTPases (Yang et al., 1998; Matern et al., 2000). More recent studies using both two-hybrid and biochemical approaches have suggested Yip1p and Yif1p are able to bind Rab proteins in a more promiscuous manner (Calero et al., 2002; Calero et al., 2003). The interaction of Yip1p with Rabs in the two-hybrid system is dependent on an intact double prenylation motif contained at the COOH termini of Rabs (Calero et al., 2003). Our study of Yip1p function revealed a critical role for Yip1p in the biogenesis of COPII transport vesicles from ER membranes (Heidtman et al., 2003). Because Ypt1p does not seem to be required for COPII vesicle budding in vivo and in vitro (Becker et al., 1991; Segev, 1991; Rexach and Schekman, 1991; Cao and Barlowe, 2000), these data suggest that Yip1p function may be independent of Ypt1p function. It is also possible that Yip1p has dual functions in both COPII vesicle formation and in modulation of Rab activity during membrane fusion (Chen et al., 2004).
In this report, we used a multicopy suppressor screen to identify genes that could rescue the thermosensitive growth defect of the yip1-4 strain. The yip1-4 allele results from an E70K point mutation in the hydrophilic NH2 terminus of Yip1p (Calero et al., 2003). This strain exhibits a severe growth defect at the restrictive temperature of 34°C on rich media with few revertants, making this strain ideal for genetic studies. Our screen identified the uncharacterized ORF YER074W-A as a novel multicopy suppressor of the yip1-4 allele. We have termed this gene YOS1. Interestingly, multicopy YOS1 is unable to suppress the previously described thermosensitive yip1-1, yip1-2, yif1-2, and yif1-4 strains (Yang et al., 1998; Matern et al., 2000).
YOS1 encodes a conserved protein of 87 amino acids with a predicted molecular mass of 9.5 kDa. We show that YOS1 is an essential gene in yeast, and Yos1p depletion results in the accumulation of the proforms of secretory proteins. In addition, we show that Yos1p localizes to ER and Golgi membranes as well as ER-derived COPII transport vesicles. This pattern of localization is very similar to that of Yip1p and indicates that Yos1p cycles between ER and Golgi compartments. Importantly, we also demonstrate that Yos1p physically associates with Yip1p and Yif1p, indicating that Yos1p is a novel subunit of the Yip1p–Yif1p complex. This interaction was disrupted in the yip1-4 strain, suggesting the NH2 terminus of Yip1p may in some way regulate the interaction of Yip1p with Yos1p.
Our previous studies indicated that inhibition of Yip1p function blocks the formation of COPII vesicles from ER membranes. Consistent with these observations, pulse-chase analysis of the thermosensitive yos1-1 strain showed a severe block in the transport of CPY at the restrictive temperature. Furthermore, membranes prepared from the yos1-1 strain exhibited transport defects most specific to the COPII budding stage in a cell-free transport assay. EM analysis of the yos1-1 strain at the restrictive temperature revealed tubulation and dislocation of ER membranes from the cell periphery. In addition, accumulation of vesicular structures or tubular elements was sometimes observed. These structures may represent transport vesicles or fragmented ER membranes. These phenotypes are similar to those reported for the yif1-1 thermosensitive strain (Matern et al., 2000). Interestingly, these morphological phenotypes are somewhat distinct from that of the yip1-4 strain at the restrictive temperature, which displays massive proliferation of ER membranes but no accumulation of vesicles (Heidtman et al., 2003). These observations may indicate that Yif1p and Yos1p function downstream of Yip1p to fulfill a critical function at the Golgi complex. Alternatively, the moderate accumulation of vesicular structures seen in the yif1-1 and yos1-1 strains may indicate an additional role for these proteins in retrograde transport. Thermosensitive mutations in genes involved in retrograde trafficking have been reported to result in an intermediate vesicle accumulation phenotype (Kaiser and Schekman, 1990; Sweet and Pelham, 1993).
The thermosensitivity of the yos1-1 strain was caused by an N18A point mutation in the first predicted transmembrane domain of Yos1p. Western blot analysis of this strain revealed that Yos1-1p was stably expressed at both the permissive and restrictive temperatures (our unpublished data). These observations suggest that this mutant form of Yos1p is stably integrated into membranes but is defective in some other aspect of protein function. One possibility is that the N18A point mutation prevents efficient interaction of Yos1p with Yip1p–Yif1p via their respective transmembrane domains. In support of this idea, we observed that Yip1p interaction with Yos1p was disrupted in the yos1-1 strain.
What is the nature of the Yos1p–Yip1p–Yif1p complex in terms of assembly and stability? To address this question, it may be useful to consider other characterized heteromeric protein complexes localized to the early secretory pathway. For example, yeast oligosaccharyltransferase (OST) is a conserved heteromeric complex that catalyzes the initial stages of N-linked protein glycosylation in the lumen of the ER (Dempski and Imperiali, 2002). Mutation in certain subunits of OST results in decreased stability of other associated subunits, indicating that at least some of the OST subunits depend on each other for stability (Silberstein et al., 1995). It is unclear whether individual subunits of the Yos1p–Yip1p–Yif1p complex are directly required for stability of other subunits. A recent study showed that GAL1 regulated depletion of Yip1p from cells resulted in decreased levels of Yif1p (Barrowman et al., 2003); however, this may be due to a general secretory block. We have observed that a reduction of Yip1p or Yif1p by using the conditional yip1-1, yip1-2, yif1-2, or yif1-4 alleles (Yang et al., 1998; Matern et al., 2000) did not produce a decreased level of the reciprocal subunit (our unpublished data). This result suggests that the expression levels of Yos1p, Yip1p, and Yif1p are not interdependent. Moreover, we find that Yos1p, Yip1p and Yif1p can be individually overexpressed severalfold (our unpublished data), supporting the idea that subunits of the complex do not depend on each other for stability. These expression behaviors are more reminiscent of SNARE protein complexes in which subunits of the SNARE complex undergo cycles of assembly and disassembly in catalysis of membrane fusion (Bonifacino and Glick, 2004). Therefore, the Yos1p–Yip1p–Yif1p complex may cycle through assembly/disassembly states in temporal and spatial alignment with biochemical reactions required for ER/Golgi transport.
Interestingly, Yos1p interaction with Yip1p is disrupted in the yip1-4 strain. The thermosensitivity of the yip1-4 strain is the result of an E70K point mutation in the cytosolic NH2 terminus of Yip1p. Therefore, the NH2 terminus of Yip1p may regulate the association of Yip1p with Yos1p, perhaps in response to a signal or factors present in the cytosol. This idea may provide an explanation for the finding that Yos1p is a multicopy suppressor of the yip1-4 strain. Specifically, overexpression of Yos1p may stimulate complex assembly in the absence of a proper signal, thus providing a minimal level of complex to allow growth at the restrictive temperature. Biochemical purification of the Yos1p–Yip1p–Yif1p complex will be required to determine composition, stoichiometry, and size of the complex.
What is the function of the Yos1p–Yip1p–Yif1p complex in the cell? The Yip1p–Yif1p complex was originally proposed to function as a receptor for the recruitment of Ypt1p to Golgi membranes (Yang et al., 1998; Matern et al., 2000). A recent study provided evidence of this type of activity for the human protein PRA-1, the human homologue of the yeast protein Yip3p (Sivars et al., 2003). In this study, the authors showed that PRA-1/Yip3 acts catalytically to dissociate complexes of endosomal Rab9 bound to GDI for Rab9 recruitment onto membranes. Although Yip3p shares a similar predicted membrane topology with Yip1p, Yip3p has a limited primary sequence relationship to Yip1p or Yif1p and therefore may be a member of a separate family of factors that act in intracellular trafficking. Depletion of Yip1p from yeast cells did not seem to effect the membrane binding or localization of Ypt1p (Barrowman et al., 2003), although the subcellular distribution of GFP-Ypt1p was altered in yip1-4 strains at a restrictive temperature (Calero et al., 2003). In light of these observations, it is possible that the action of the Yos1p–Yip1p–Yif1p complex may be restricted to the budding stage of transport. A cycling heteromeric complex of Yos1p–Yip1p–Yif1p could act at both ER and Golgi membranes to promote COPII and COPI vesicle biogenesis, perhaps in promoting vesicle scission. Alternatively, Yip1p and associated proteins may interact with Ypt1p and other Rab GTPases to coordinate the budding and tethering stages of intracellular transport. Identification of Yos1p as a new member of the Yip1p–Yif1p complex will allow us to perform more defined biochemical and morphological experiments to understand the precise function of the Yos1p–Yip1p–Yif1p complex in eukaryotic cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Address correspondence to: Charles Barlowe (barlowe{at}dartmouth.edu).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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