Correspondence to: M. Gerard Waters, Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Tel:(609) 258-2891 Fax:(609) 258-1701 E-mail:gwaters{at}molbio.princeton.edu.
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Abstract |
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A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ~750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.
Key Words: Sec34p, Sec35p, Rud3p, vesicle tethering, secretory pathway
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Introduction |
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THE flow of material through the secretory pathway is mediated, at least in part, by membrane-bound vesicles or larger membrane-delimited structures (
For some time, members of the rab GTPase family were suspected to be the principle determinants of this targeting specificity because distinct family members display unique organellar localizations that correlate with their site of action (for review see
Given that neither rabs nor SNAREs are the sole governors of targeting specificity, other components are likely to play important roles. Additional candidate players include the so-called tethering factors (
ER to Golgi complex traffic in the yeast Saccharomyces cerevisiae is one of the most intensively studied steps in membrane trafficking. The components involved in the consumption of ER-derived vesicles at the Golgi complex are related to those of many other steps. These include: the v-SNAREs Bet1p, Bos1p, Sec22p, and perhaps Ykt6p (
Two of the accessory proteins required for ER to Golgi complex vesicle transport in yeast are Uso1p (
In addition to Uso1p and Sec35p, another component that may function in the tethering of ER-derived vesicles is an ~800-kD protein complex with ten subunits, termed TRAPP (
In an effort to further elucidate the mechanism of vesicle tethering, we have studied SEC34. sec34 mutants were identified in a novel screen for secretion mutants in the early secretory pathway of S. cerevisiae and were shown to block ER to Golgi complex traffic concomitant with an accumulation of transport vesicles (
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Materials and Methods |
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Media and Microbial Techniques
Bacterial media was prepared by standard protocols (
Plasmid and Strain Construction
Plasmids used in this work are described in Table 1. Plasmid construction was as follows. To generate pSV22, the genomic library plasmid pB4 was digested with PvuII and HindIII, and the resulting 2.7-kb fragment containing YER157w/SEC34 was ligated into pRS416 that had been digested with SmaI and HindIII. To create pSV24 and pSV25, SEC34 was liberated from the polylinker of pSV22 with either HindIII and BamHI or XhoI and SpeI double-digests and ligated into pRS305 (digested with HindIII and BamHI) or pRS426 (digested with XhoI and SpeI), respectively. The insert for bacterial expression plasmids encoding glutathione S-transferase (GST)-Sec34p and His6-Sec34p fusion proteins was generated by PCR including a BamHI site adjacent to the codon for the first amino acid of Sec34p and a SmaI site downstream of the stop codon (5' primer, 5' gcc-gga-tcc-atg-gcg-aga-agt-aga-aag 3'; 3' primer, 5' tcc-ccc-ggg-gtt-tat-ttc-gtt-atg-gta-tc 3'). The PCR product was digested with BamHI and SmaI, and ligated into similarly digested pGEX4T-1 (Pharmacia Biotech, Inc.) and pQE30 (QIAGEN, Inc.), generating pSV28 and pSV30, respectively. To create the constructs expressing the Gal4p DNA binding domain (Gal4p-BD) or transcriptional activation domain (Gal4p-AD) fused to Sec34p, the SEC34 open reading frame (ORF) was amplified by PCR, placing a BamHI site upstream of the codon for the second amino acid residue of the protein and a PstI site downstream of the stop codon (5' primer, 5' cgc-gga-tcc-tgg-cga-gaa-gta-gaa-ag 3'; 3' primer, 5' cgc-gct-gca-gtt-tat-ttc-gtt-atg-gta-tc 3'). The resulting product was cleaved with BamHI and PstI, and ligated into a similarly digested pAS2 or pGAD424 (Clonetech, Inc.), yielding COOH-terminal fusions to Gal4p-BD (pSV37) and Gal4p-AD (pSV35), respectively. The constructs expressing the Gal4p-BD or Gal4p-AD fused to Sec35p were constructed in an identical manner, also placing a BamHI site upstream of the codon for the second amino acid residue of the protein and a PstI site downstream of the stop codon (5' primer, 5' cgc-gga-tcc-tgg-tca-aca-gtc-ata-g 3'; 3' primer, 5' cgc-gct-gca-ggt-ttt-ctc-cca-act-atg 3'), creating COOH-terminal fusions to Gal4p-BD (pSV34) and Gal4p-AD (pSV36), respectively. To delete one copy of SEC34 in a diploid strain by the -method (
plasmid, pSV27, was constructed in two stages. In the first stage, the region 5' to SEC34 was excised from plasmid pB4 as a PstI/PvuII fragment (the location of restriction enzymes sites are shown in Figure 1 a) and ligated into PstI/SmaI-digested pRS305. In the second stage, a HindIII fragment containing the region 3' to the locus was released from plasmid pB4 and ligated into the plasmid generated from step one, which had been linearized with HindIII; the correct orientation of the HindIII fragment was confirmed by restriction digest. In the resulting construct, the inserts in the polylinker of pRS305 were placed such that the region directly 3' to the ORF was placed upstream of the region directly 5' of the ORF, with a unique restriction site, PstI, between the two sequences; digestion of this plasmid with PstI and transformation of the linearized plasmid into a diploid strain results in the replacement of the coding sequences of one allele of SEC34 with the sequences of the integrating vector. To generate pSK81, ORF YOR216c/RUD3 was isolated from the library plasmid pSOU7 (
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Yeast strains used in this paper are described in Table 2. Strain construction was as follows. The sec34::LEU2/SEC34 strain GWY127 was constructed by transforming GWY30 with PstI-digested pSV27. The presence of the SEC34 deletion in the Leu+ transformants was confirmed by PCR amplification of the novel junctions at the deletion locus. The diploid strain heterozygous for both the sec34-2 and sec35-1 alleles was created by mating GWY93 to GWY95 for 6 h on YPD. The diploid was identified by the distinct morphology of the zygote, and was isolated by micromanipulation.
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Cloning of SEC34
To clone SEC34, the sec34-2 strain GWY95 (
Antibody Production and Immunoblotting
GST-Sec34p and His6-Sec34p were expressed in strain XL1-Blue (Stratagene) from plasmids pSV28 and pSV30, respectively, and fusion proteins were purified according to the manufacturers' instructions (Pharmacia Biotech, Inc.; QIAGEN, Inc.). GST-Sec34p was used to immunize rabbits by standard procedures (
Extraction and Subcellular Fractionation
A 250-ml culture of wild-type yeast (RSY255) was grown in YPD at 30°C to midlogarithmic phase (OD595 = 1.4), washed in sterile water, and resuspended at ~75 OD595/ml in Buffer 88 (25 mM Hepes, pH 7.0, 150 mM KOAc, 5 mM MgCl2, 1 mM DTT) containing protease inhibitors (
In Vitro ER to Golgi Complex Transport Assay
Yeast semi-intact cells from either the wild-type (RSY255) or the sec34-2 strain (GWY95) were prepared from logarithmic phase cultures of strains grown at 23°C and were stored at -70°C (-factor was posttranslationally translocated into the ER of the semi-intact cells as previously described (
-factor (35S-gp-
-factor) contained in these vesicles was quantified after solubilization of membranes and precipitation with Concanavalin ASepharose. For transport assays, COPII proteins, in addition to purified Uso1p and LMA1, were added as indicated in the figure legends, and the amount of Golgi complex-modified 35S-gp-
-factor was measured by immunoprecipitation with anti-
1,6-mannosespecific antibodies. The data presented is the average of duplicate determinations and the error bars represent the range.
Partial Purification of the Sec34p/Sec35p Complex
The protease deficient RSY1157 strain was grown to late log phase (OD600 = 3.4) in 36 liters of YPD at 30°C, after which all manipulations were performed at 04°C. The cells were harvested by centrifugation and washed twice with water. The 334 g cell pellet was resuspended in 1 liter of 25 mM Tris-Cl, pH 8.0, 1 M KCl, 2 mM EGTA (lysis buffer) with protease inhibitors [0.5 mM 1:10 phenanthroline, 2 µM pepstatin A, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 mM PMSF, 200 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)] and 1 mM DTT, and lysed in an EmulsiFlex-C5 (Avestin Inc.) at 18,00020,000 psi. The lysate was centrifuged at 5,000 g (Sorvall SLA3000 rotor, 6,000 rpm, 10 min), and the supernatant (1.04 liter) was collected and centrifuged at 20,000 g (Sorvall SA600 rotor, 12,000 rpm, 20 min). The supernatant (960 ml) was removed, avoiding the loose pellet, and centrifuged at 175,000 g (Beckman 45Ti rotor, 44,000 rpm, 120 min). The supernatant (S175; 750 ml at 7.3 mg protein/ml) was removed, avoiding the pellets. The S175 was made 1 mM in EDTA, and (NH4)2SO4 was added to 35% saturation, dissolved, and the solution was stirred for 60 min. The (NH4)2SO4 precipitate was collected by centrifugation at 17,000 g (SLA3000 rotor, 10,000 rpm, 10 min), resuspended in enough 25 mM Tris-Cl, pH 8.0, 1 mM DTT to yield a conductivity equivalent to that of 25 mM Tris-Cl, pH 8.0, 100 mM KCl, 1 mM DTT (T8.0/100K/D). This material (1.55 g protein in 368 ml; 4.2 mg/ml) was loaded at 2 ml/min onto a 50 ml DEAE-Sepharose Fast Flow (Pharmacia Biotech, Inc.) column (2.5 cm i.d.) that had been equilibrated in T8.0/100K/D. The column was washed with 200 ml of T8.0/100K/D and eluted with a linear gradient (2 mM/ml) from T8.0/100K/D to T8.0/400K/D, collecting 10 ml fractions throughout. At this and each subsequent chromatographic step, fractions were analyzed for Sec34p and Sec35p content by immunoblotting. The fractions containing Sec34p and Sec35p, which eluted together at ~160 mM KCl, were pooled (348 mg protein in 60 ml, 5.8 mg/ml). A 200 µl aliquot of this DEAE pool was chromatographed on a 24 ml Superose 6 (HR10/30 Pharmacia Biotech, Inc.) size exclusion column equilibrated in T8.0/150K/D at 0.3 ml/min, collecting 1 ml fractions. The remaining 60 ml of the DEAE pool was then concentrated to a volume of 17.8 ml (284 mg protein at 16 mg/ml) in an Amicon 8050 Ultrafiltration Cell (Millipore Inc.) with a YM100 (MWCO 100 kD) ultrafiltration membrane. The concentrated sample was loaded onto a 700 ml Sephacryl S-300 (Pharmacia Biotech, Inc.) column (2.5 cm i.d.) that had been equilibrated in 25 mM Tris-Cl, pH 7.6, 100 mM KCl, 1 mM DTT (T7.6/100K/D) and chromatographed at 1.5 ml/min in T7.6/100K/D, collecting 10 ml fractions. The fractions containing both Sec34p and Sec35p were pooled (66 mg protein in 47 ml, 1.4 mg/ml) and loaded onto an 8 ml MonoQ (HR 10/10, Pharmacia Biotech, Inc.) anion exchange column equilibrated in T7.6/100K/D at 1.5 ml/min. The column was washed with 36 ml of T7.6/100K/D and eluted with a linear gradient (2.5 mM/ml) from T7.6/100K/D to T7.6/500K/D, collecting 5 ml fractions. The fractions containing both Sec34p and Sec35p, which coeluted at ~295 mM KCl, were pooled (16 mg protein in 18 ml, 0.89 mg/ml), dialyzed against 40 mM potassium phosphate, pH 6.8, 0 mM KCl, 1 mM DTT (KP/0K/D), and loaded onto a MonoS (HR 5/5, Pharmacia Biotech, Inc.) equilibrated in KP/0K/D at 0.5 ml/min. The column was washed with 4 ml of KP/0K/D and eluted with a linear KCl gradient (10 mM/ml) from KP/0K/D to KP/500K/D, collecting 2 ml fractions throughout. The fractions containing both Sec34p and Sec35p, which coeluted at ~110 mM KCl, were pooled (1.5 mg protein in 8 ml, 0.19 mg/ml) and concentrated to a volume of 0.1 ml at 9.3 mg/ml with an Ultrafree BIOMAX centrifugal device (Millipore Inc.). The concentrated sample was loaded onto a 24 ml Superose 6 (HR10/30, Pharmacia Biotech, Inc.) size exclusion column that had been equilibrated in T7.6/200K/D, and chromatographed at 0.3 ml/min, collecting 0.5 ml fractions. After this point, protein concentration was no longer determined, to not decrease yield. The fractions containing Sec34p and Sec35p were pooled and loaded onto a Bio-Scale CHT2-I Hydroxyapatite column (Bio-Rad Laboratories). The column was washed with 8 ml 25 mM Tris-Cl, pH 8.0, 200 mM KCl, 0 mM potassium phosphate, 1 mM DTT (T7.6/200K/0Pi/D), and eluted with a linear potassium phosphate gradient (16.6 mM/ml) from T7.6/200K/0Pi/D to T7.6/200K/250Pi/D; Sec34p and Sec35p coeluted at ~100 mM potassium phosphate.
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Results |
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Cloning of SEC34
To clone SEC34, the sec34-2 strain was transformed with a low-copy (centromere, CEN) yeast genomic library and temperature-resistant colonies were selected at 38.5°C. Library plasmids were isolated from these colonies and retested for their ability to confer growth at 38.5°C. Four restored growth at the restrictive temperature to wild-type levels, and the remaining three yielded partial suppression. The ends of the inserts of the seven library plasmids were sequenced and found to contain overlapping regions of the right arm of chromosome V, a portion of which is shown in Figure 1 a. The only complete ORF contained on each of the plasmids that conferred strong suppression of the temperature-sensitive phenotype (two of which are shown in Figure 1b and Figure c) was YER157w. Interestingly, the three plasmids that partially suppress the sec34-2 mutation contained identical inserts in which only the 5' end of YER157w is present; the inability of these plasmids to fully suppress may be due to the absence of the COOH-terminal portion of the protein. The ORF YER157w was isolated from the genomic insert (as shown in Figure 1 d) and transferred to a low-copy plasmid. This construct was demonstrated to suppress the temperature sensitivity of the sec34-2 strain, confirming that YER157w is responsible for the suppression conferred by each of the library plasmids. To address the possibility that YER157w was a suppressor of the sec34-2 mutation rather than the gene itself, integrative mapping was performed. A sec34-2 strain in which the YER157w locus was marked with LEU2 was constructed and subsequently mated to a wild-type strain. The resulting diploid strain was subjected to tetrad analysis and, of 38 tetrads examined, the temperature-sensitive phenotype did not segregate away from the marked locus. Thus, integrative mapping strongly suggests that YER157w is SEC34.
SEC34 is predicted to encode an 801-amino acid protein (Figure 1 f) with a molecular weight of 92.5 kD and a pI of 5.2. Sec34p lacks a signal sequence, as well as transmembrane domains or other motifs that could facilitate membrane attachment. Therefore, the protein is predicted to be either cytoplasmic or peripherally associated with membranes. Three putative orthologs of Sec34p have been detected. First, the C. elegans genome encodes a protein designated Y71F9A 290.A that is 25% identical and 35% similar to Sec34p. This 428-amino acid protein is ~50% the size of Sec34p and is homologous to the NH2 terminus of Sec34p (spanning amino acid residues 111 to 459). Due to the disparity in size, it is unclear whether this protein is indeed an ortholog of Sec34p. Second, the genome of the fission yeast Schizosaccharomyces pombe contains a 735-amino acid protein (GenBank/EMBL/DDBJ accession number CAB51337, PID g5579050) that is 26% identical and 40% similar to Sec34p. Finally, several overlapping human expressed sequence tags (ESTs; GenBank/EMBL/DDBJ accession numbers AA280321, AA603511, AA429818, and z21241) have been isolated that show high similarity to Sec34p. Sequencing of clones containing the first two ESTs (provided by Genome Systems, Inc., St. Louis, MO) allowed us to analyze additional sequences previously unavailable in GenBank (data not shown). By combining our newly sequenced regions with the overlapping ESTs in the database, we obtained a 263-amino acid portion of the putative human protein. Comparison of this partial protein to Sec34p using the BLAST algorithm (
The SEC34 Deletion Strain Displays a Severe Growth Defect
To evaluate the phenotype of a strain lacking SEC34, we constructed a diploid strain in which one allele of SEC34 had been deleted (as diagrammed in Figure 1 e) and replaced with the gene LEU2. This strain (sec34::LEU2/SEC34) was sporulated and dissected, and the resulting tetrads were incubated on rich media at 30°C. As shown in Figure 2 (left), a clear 2+:2- segregation pattern was observed in which each tetrad contained two large and two very small colonies. The large colonies were without exception Leu- and thus contained the wild-type copy of the gene, while the small colonies were Leu+, indicating the presence of the sec34
locus. Therefore, although SEC34 is not an essential gene, haploid strains lacking SEC34 are at an extreme growth disadvantage. The growth defect of the sec34
strain is complemented by a plasmid bearing SEC34 since the presence of the construct in the diploid sec34
/SEC34 strain resulted in the restoration of a 4+:0- segregation pattern (Figure 2, right). In each tetrad, two segregants were Leu+ and Ura+, indicating the presence of both the deletion and the plasmid bearing SEC34, respectively.
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Genetic Analysis of SEC34
Previous analysis of the sec34 mutant strain indicated a role for Sec34p in the docking or fusion of ER-derived vesicles with the cis-Golgi complex (
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Several other genes were tested for their ability to suppress the sec34-2 mutation when overexpressed, but were found to have no effect. These genes encode the tethering factor Sec35p, the TRAPP complex component Bet3p, the cis-Golgi complex t-SNARE Sed5p, and the Golgi complex to plasma membrane rab protein Sec4p. The lack of suppression by the Golgi complex to plasma membrane rab indicates that suppression by the ER to Golgi complex rab is specific.
We also tested whether overexpression of SEC34 could suppress temperature-sensitive mutant alleles of several ER to Golgi complex docking factors. Interestingly, overexpression of SEC34 was capable of weakly suppressing the temperature-sensitive growth defect of the sec35-1 strain (data not shown). However, multicopy SEC34 was unable to suppress mutant alleles of all other secretory factors tested, including: the v-SNAREs Sec22p, Bos1p, and Bet1p; the t-SNARE Sed5p; the tethering factor Uso1p; the rab Ypt1p; and the TRAPP complex component Bet3p (data not shown). Therefore, overexpression of either SEC34 or SEC35 (
Since YPT1 and SLY1-20 were efficient suppressors of the temperature-sensitive growth defect of the sec34-2 strain, we explored whether they could also improve the severe growth defect of the sec34 strain. The sec34
/SEC34 diploid strain was therefore transformed with plasmids expressing either multicopy YPT1 or low-copy SLY1-20, and the resulting diploids were sporulated and subjected to tetrad analysis. Indeed, the presence of either plasmid significantly improved the growth of the sec34
strain (Figure 2, right). This result implies that Ypt1p and Sly1p most likely function downstream of, or in a parallel pathway with, Sec34p.
Isolation of RUD3 and its Genetic Interactions with SEC34 and USO1
Suppression of the sec34-2 temperature-sensitive growth defect was also observed upon overexpression of a gene designated RUD3 (Figure 3). RUD3 was isolated previously in our lab through a screen for genes that, when overexpressed, are able to suppress the temperature-sensitive growth defect of the uso1-1 strain (
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RUD3 is predicted to encode a 484-amino acid protein with a molecular mass of 56.1 kD and a pI of 4.67 (Figure 4 b). Like Sec34p, this protein lacks any motifs to indicate a localization other than cytosolic. Both the PAIRCOIL and COILS programs (
To test whether Rud3p is encoded by an essential gene, a diploid strain was created in which one of the alleles of RUD3 was deleted and marked with LEU2. Sporulation and tetrad dissection of this strain yielded tetrads with four viable spores, two of which were Leu+. Upon incubation at 25, 30, and 37°C, the haploid rud3 strain did not display a significant growth defect as compared with a wild-type strain (data not shown), and thus RUD3 is not an essential gene.
Sec34p Is a Peripheral Membrane Protein
To analyze the Sec34 protein, we generated affinity-purified anti-Sec34p antibody. The antibody recognizes two proteins in crude yeast extracts, the larger of which corresponds to the predicted molecular weight of Sec34p (Figure 5 a). This protein is absent in a sec34 strain (which expressed SLY1-20 to enhance its propagation) and is overexpressed in a strain containing SEC34 on a multicopy plasmid, and thus represents Sec34p. The smaller protein, which is recognized despite the affinity purification, is not related to the Sec34p locus since it is present in the sec34
strain and its expression level is unaffected by overexpression of SEC34.
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With this antibody, we investigated whether Sec34p was capable of associating with membranes, as might be expected for a protein involved in secretion. A crude yeast extract (designated S1) was centrifuged at 175,000 g to separate the organelles of the secretory pathway, which are found in the pellet fraction, from cytosolic proteins, which are contained in the supernatant fraction. As shown in Figure 5 b (left), the majority of Sec34p is found in the pellet fraction, along with the integral membrane protein Sed5p, while a small amount of Sec34p is contained in the supernatant, as is the cytosolic marker, PGK. This result is consistent with a peripheral membrane association for Sec34p. To explore the basis for the sedimentation of Sec34p, we attempted to extract the protein from an enriched membrane fraction using buffers containing Triton X-100, NaCl, or Na2CO3, pH 11.5. As expected for an integral membrane protein, Sed5p was released into the supernatant fraction after incubation with buffer containing Triton X-100, but not after treatment with salt or high pH, whereas the peripheral membrane protein Sec35p was released, at least partially, upon incubation with all three buffers. Sec34p was partially shifted into the supernatant fraction upon treatment with Triton X-100, salt, or high pH, and thus behaves as a peripheral membrane protein (Figure 5 c).
To further analyze the membrane association of Sec34p, differential centrifugation was employed. The S1 fraction was centrifuged at 10,000 g, separated into supernatant (S10) and pellet (P10) fractions, and the S10 fraction was further centrifuged at 175,000 g and separated into supernatant (S175) and pellet (P175) fractions. Under these conditions, the ER is contained primarily into the P10 fraction (data not shown), the Golgi complex partitions between the P10 and P175 fractions, as seen with the Golgi protein Sed5p, and cytosolic proteins remain in the supernatant fractions, as observed for PGK (Figure 5 b, right). The soluble portion of Sec34p is found in the S175 fraction, while the membrane-associated pool partitions between the P10 and P175, similar to the Golgi complex protein Sed5p. The fractionation pattern of Sec34p is quite similar to that observed for the peripheral membrane protein Sec35p, although Sec34p appears to have a greater proportion of the protein in the membrane fractions.
Sec34p Is Required for Tethering of ER-derived Vesicles to the cis-Golgi Complex
Because the mutant phenotype (
In the first assay (Figure 6 a), overall ER to Golgi complex transport is measured in semi-intact cells incubated with a mixture of purified protein components that drive all the stages of transport: vesicle formation from the ER is supported by the addition of COPII proteins, efficient vesicle tethering requires added Uso1p, and vesicle fusion requires a protein complex termed LMA1. Productive transport is monitored by following the addition of 1,6-mannose residues to gp-
-factor, an event that occurs in the cis-Golgi complex. Generation of this transport system from conditional mutants has shown that the system also requires the activities of several peripheral and integral membrane proteins including Ypt1p, Sec35p, and the SNAREs (
-factor from the ER to the Golgi complex in this in vitro system proceeds with a similar efficiency (Figure 6 a). In contrast, at 29°C in sec34-2 semi-intact cells, this process is very inefficient relative to wild-type, indicating that sec34-2 is defective for overall ER to Golgi transport in vitro.
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The second assay we employed examined the functionality of the vesicle budding and vesicle tethering steps in the sec34-2 mutant (Figure 6 b). In this assay, release of vesicles from semi-intact cells is detected by the appearance of protease-protected gp--factor in a low-speed supernatant at the end of the reaction. Vesicles were efficiently generated upon addition of COPII components to wild-type or sec34-2 semi-intact cells at 23 or 29°C (Figure 6 b). These data indicate that Sec34p is not required for ER-derived vesicle budding. Because addition of Uso1p significantly reduced vesicle release, vesicle tethering was also functional in the semi-intact wild-type cells at 23 or 29°C, as well as in sec34-2 semi-intact cells at 23°C. In contrast, when Uso1p was added to the sec34-2-derived system at the restrictive temperature of 29°C, vesicle release was only slightly diminished (Figure 6 b). This result indicates that the sec34-2 mutant cannot efficiently tether ER-derived vesicles to the yeast Golgi complex.
sec34 and sec35 Display a Synthetic Lethal Interaction
The similar genetic interactions of SEC34 and SEC35 with genes involved in the docking stage of vesicular transport, taken together with their genetic interaction with one another, and with the finding that both proteins function in vesicle tethering, lead us to examine whether mutations in the two genes would display a synthetic lethal interaction. To do this, we generated a diploid strain heterozygous for both the sec34-2 and sec35-1 alleles and subjected it to tetrad analysis. Although both the sec34-2 and sec35-1 haploid strains are permissive for growth at both 21 and 30°C, tetrads from the diploid sec34-2/SEC34 SEC35/sec35-1 strain yielded numerous inviable colonies at either temperature (Figure 7 a). After incubation of the segregants for long periods of time, a small proportion of those previously characterized as inviable would form visible microcolonies. Since this phenotype was variable, we hypothesize that the microcolonies result from either the appearance of spontaneous suppressors of the inviability or from background mutations in the strain. Examination of the viable segregants in each tetrad for temperature sensitivity revealed a pattern in which the inviable segregants are predicted to be those containing both the sec34-2 and the sec35-1 alleles. To confirm this prediction, the diploid strain was transformed with low-copy plasmids bearing either SEC34 or SEC35 before tetrad dissection. The presence of either plasmid lead to a greater proportion of viable segregants than was observed for the untransformed strain, concurrent with the appearance of segregants that were sensitive to the drug 5-fluoro-orotic acid, which is toxic to cells that must maintain the plasmid to survive (data not shown). Therefore, the sec34-2 and sec35-1 alleles display a synthetic lethal phenotype, which can be complemented by the presence of either gene on a plasmid.
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Sec34p and Sec35p Interact in the Two-hybrid Assay
In many cases, synthetic lethality between alleles of two genes involved in secretion indicates that their gene products are involved in the same stage of secretion ( and ß subunits of tubulin (
Sec34p and Sec35p Are Components of a Large Protein Complex
To characterize the interaction of Sec34p and Sec35p further, we employed immunoblotting to monitor the behavior of these proteins during fractionation of yeast cytosol. Sec34p and Sec35p coprecipitated in 35% saturated ammonium sulfate and cofractionated precisely by DEAE anion exchange chromatography (data not shown). An aliquot of the Sec34p/Sec35p anion exchange pool was then subjected to size exclusion chromatography on Superose 6 (Figure 8 a), and once again, Sec34p and Sec35p precisely cofractionate. Interestingly, they elute from the column slightly before thyroglobulin, a 669-kD globular protein. These results are consistent with Sec34p and Sec35p existing in a large protein complex with a mass of up to ~750 kD. A small amount of monomeric Sec35p is also evident upon gel filtration, suggesting either that some Sec35p has dissociated from the complex or that a cytosolic pool of monomeric Sec35p exists. No such monomeric Sec34p has been detected in cytosolic fractions. To further purify the Sec34p/Sec35p complex, the remainder of the DEAE anion exchange pool was subjected to several sequential chromatographic steps (see Materials and Methods), including Sephacryl S-300 gel filtration (data not shown), MonoQ anion exchange (Figure 8 b), MonoS cation exchange (Figure 8 c), Superose 6 gel filtration (data not shown), and ceramic hydroxyapatite (Figure 8 d). Once again, Sec34p and Sec35p precisely comigrate through each step, strongly indicating that Sec34p and Sec35p are present in a large protein complex.
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Discussion |
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Much effort has been extended towards gaining an understanding of the mechanism of transport vesicle docking in the secretory pathway. Several families of proteins are involved in this event, including the rab family of small GTP-binding proteins and the SNARE family of integral membrane proteins. Recently, another class of proteins has been described, the tethering factors. Although these proteins do not display homology with one another and thus do not define a family, they share a similar function in docking, that of connecting the vesicle to the target compartment before the interaction of v- and t-SNAREs (for reviews see
A recent genetic screen identified temperature-sensitive alleles of two genes, SEC34 and SEC35, that, when incubated at the restrictive temperature, are defective in ER to Golgi complex transport and accumulate large numbers of vesicles (
To begin our study of SEC34, we cloned the gene by complementation of the temperature-sensitive phenotype of a strain bearing the sec34-2 mutation. SEC34 was discovered to be a novel gene encoding a protein with a predicted molecular weight of 93 kD. Deletion of SEC34 in a haploid strain resulted in a severe growth defect, and thus SEC34 is essential for wild-type growth rates, although not for viability.
To investigate the genetic interactions of SEC34 we employed multicopy suppressor analysis. The best suppression of the sec34-2 temperature-sensitive growth defect was conferred by overexpression of Ypt1p, the rab required in ER to Golgi complex transport, or by expression of Sly1-20p, the dominant form of the t-SNAREassociated factor, Sly1p. Suppression of the SEC34 deletion strain allowed us to order the action of Sec34p with respect to Ypt1p and Sly1p. Since either YPT1 or SLY1-20 can suppress both mutations in, and a deletion of, SEC34, yet overexpression of SEC34 cannot suppress mutations in either YPT1 or SLY1, we hypothesize that Ypt1p and Sly1p function downstream of Sec34p.
Weaker suppression of the sec34-2 mutation was observed upon overexpression of the tethering factor Uso1p, or the v-SNAREs Sec22p, Bet1p, or Ykt6p. The suppression of sec34-2 by the v-SNAREs may be through mass action, in which vesicles containing supernumerary v-SNAREs are able to compensate for a deficiency in tethering, albeit with a very low efficiency. This phenomena has been observed previously for mutations in the tethering factors Uso1p ( strain (
Biochemical analysis of the Sec34 protein reveals that it is a peripheral membrane protein. Although a small amount of the protein is soluble, the remainder partitions between the P10 and P175 fractions, similar to the Golgi protein Sed5p; it is possible, therefore, that Sec34p is associated with the Golgi complex. Due to the association of Sec34p with membranes, we used semi-intact cells made from the sec34-2 strain to test the requirement for Sec34p in tethering through an assay that reconstitutes ER to Golgi complex transport. These semi-intact cells were demonstrated to be able to bud vesicles from the ER, but these vesicles failed to efficiently tether to the Golgi complex at the restrictive temperature, indicating that Sec34p is required for the tethering of ER-derived vesicles to the cis-Golgi complex. Since cytosolic proteins are removed from the sec34-2 semi-intact cells, the membrane-associated pool of Sec34-2p is most likely the source of the tethering defect. In addition, since the membranes involved in tethering are restricted to those of the vesicle and the cis-Golgi complex, Sec34p is most likely associating with one, or both, of these membranes.
SEC34 was found to display two interesting genetic interactions with the tethering factor gene SEC35. First, multicopy SEC34 weakly suppresses a temperature-sensitive allele of SEC35. Since overexpression of SEC34 cannot suppress the cold-sensitive lethality of the sec35 strain, Sec34p is able to assist a handicapped allele of SEC35, but cannot replace its function. Second, the sec34-2 and sec35-1 alleles display a synthetic lethal interaction. Although strains bearing either allele alone are permissive for growth at 23 and 30°C, a haploid strain containing both the sec34-2 and sec35-1 alleles is inviable at either temperature. This synthetic phenotype is more severe than the conditional synthetic lethality of the sec35-1 allele in combination with a mutant allele of either YPT1 or USO1, in which the double mutants are viable at 23°C, but not at 30°C (
Based on these results, we investigated whether Sec34p and Sec35p could physically interact through the two-hybrid assay. Indeed, Sec34p and Sec35p were found to interact. The interaction between the two proteins may explain the ability of multicopy SEC34 to suppress the sec35-1 allele, but not the sec35 allele: increased levels of Sec34p could stabilize a defective form of Sec35p but would be ineffectual in the absence of Sec35p, especially if the interaction of the two proteins is essential to their function in tethering. To further explore the interaction of Sec34p and Sec35p we examined the behavior of the soluble pool of these proteins through several chromatographic steps. The proteins cofractionated through ammonium sulfate precipitation and anion exchange, cation exchange, ceramic hydroxyapatite, and size exclusion chromatographic steps, providing strong evidence that the two proteins are in a complex with one another. Intriguingly, the Sec34p/Sec35p complex appears quite large, with an estimated molecular weight (if globular) of ~750 kD. This size, which is larger than the combined molecular weights of the two proteins (124 kD), suggests several possibilities for the structure of the complex. First, the complex could be homodimeric, containing one molecule of each protein, but highly elongated such that it migrates rapidly through a size exclusion column. We consider this unlikely because the sequences of Sec34p and Sec35p lack motifs (such as coiled-coil domains) that would be indicative of an elongated structure. Second, the complex could contain two or more molecules of at least one protein, resulting in a more massive structure. Finally, the complex could be multimeric, containing heretofore unidentified component(s) in addition to Sec34p and Sec35p. We are currently purifying the Sec34p/Sec35p complex to address this issue and identify any additional components. It appears, however, that Uso1p is unlikely to be a component of the Sec34p/Sec35p complex since immunoblotting fractions from the purification with an antibody against this protein revealed that Uso1p did not comigrate with Sec34p and Sec35p (data not shown).
The 750-kD complex containing Sec34p and Sec35p is reminiscent of the TRAPP complex, which migrates at ~800 kD by size exclusion chromatography (
Since many secretory factors are evolutionarily conserved, we explored whether the components of the Sec34p/Sec35p complex were conserved in higher eukaryotes. The genome of the nematode C. elegans was discovered to contain a protein designated Y71F9A 290.A that is very similar to Sec34p. However, the C. elegans protein is ~50% the size of Sec34p and therefore may not be a true ortholog. We also discovered a C. elegans protein with moderate homology to Sec35p (22% identical and 33% similar), designated C35A5.6. While the similarity is not high, the proteins are similar in size (C35A5.6 is comprised of 273 amino acid residues, whereas Sec35p is comprised of 275 amino acid residues), and thus, this C. elegans protein is a putative ortholog of Sec35p. Searches of GenBank for additional homologs of these proteins did not reveal additional Sec35p homologs, but several human ESTs were discovered with a high degree of similarity to Sec34p. Interestingly, the sequences contained on these ESTs were homologous to Sec34p over only a portion of the analyzed region of the putative human protein, and thus the protein may contain a Sec34p-like domain and may not be a true Sec34p ortholog. These data indicate that there may be orthologs of the Sec34p/Sec35p complex in higher organisms, but functional experiments will be required to unambiguously address this point. Finally, a putative ortholog of Sec34p was discovered in the genome of S. pombe. No paralogs of either Sec34p or Sec35p exist in S. cerevisiae, and thus these proteins do not define a family of related proteins.
Finally, we describe the identification and characterization of a gene designated RUD3 that displays a genetic interaction with SEC34. RUD3, which encodes a novel nonessential protein with a predicted molecular weight of 56 kD, was originally identified in a screen for multicopy suppressors of a temperature-sensitive allele of the tethering factor, USO1 ( strain (data not shown). Taken together, these data suggest that Rud3p either acts at, or downstream of, the tethering stage of ER to Golgi complex transport. Rud3p does not appear to be a component of the Sec34p/Sec35p complex since the majority of the protein fractionates away from the complex during its purification (data not shown).
In summary, we describe the characterization of a novel secretory factor, Sec34p, and its role in tethering of ER-derived vesicles to the cis-Golgi complex. Unlike the SNAREs and rabs, the tethering factors described thus far at different intracellular transport steps are not members of a protein family. Nevertheless, they do share structural similarity, since they are either elongated or present in a large multimeric complex (
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Footnotes |
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Stephanie K. Sapperstein's present address is Department of Neurobiology, Stanford University Medical Center, Stanford, CA 94305.
Vladimir V. Lupashin's present address is Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205.
1 Abbreviations used in this paper: CEN, centromere; Gal4p-AD, Gal4p transcriptional activation domain; Gal4p-BD, Gal4p DNA-binding domain; gp--factor, glycosylated pro-
-factor; GST, glutathione S-transferase; ORF, open reading frame; PGK, phosphoglycerate kinase; SNARE, soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor; t-SNAREs, SNAREs found predominantly on target membranes; v-SNAREs, SNAREs found predominantly on vesicles.
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We thank Randy Schekman for the original sec34 and sec35 strains. We are grateful to S. Ferro-Novick, P. James, P. Novick, H. Pelham, M. Rose, R. Schekman, and members of their laboratories for generously supplying reagents and strains, and D. Hasara for expert assistance in antibody production. We thank Bryan Kraynack, Barbara Reilly, and Misha Rosenbach for assistance with the two-hybrid assay.
This work was supported by grants from the American Cancer Society (RPG-98-050-01-CSM to M.G. Waters), the National Institutes of Health (GM52549 to C. Barlowe), the Pew Scholars Program in the Biomedical Science (C. Barlowe), and a fellowship from the Lucille P. Markey Charitable Trust (M.G. Waters). S.M. VanRheenen and E.C. Chiang were supported by a National Institutes of Health training grant (GM07312) and S.K. Sapperstein was supported by an American Heart Association predoctoral fellowship.
Submitted: 27 July 1999
Revised: 6 October 1999
Accepted: 12 October 1999
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