sec36-1 Mutation Blocks Transport of Several Proteins through the Secretory Pathway
In a genetic screen for mutants defective in ribosome synthesis, temperature-sensitive strains were isolated that seemed to be primarily impaired in transport of secretory proteins (Mizuta and Warner, 1994
; Li and Warner, 1996
). One of these mutants, 271ts, showed accumulation of the secreted protein CPY in its ER-modified p1 form at 37°C, indicating a defect in transport between the ER and medial Golgi compartment (Li and Warner, 1996
). When strain 271ts was backcrossed four times to the S288C strain background, temperature sensitivity and the CPY transport defect cosegregated as a single nuclear gene mutation (our unpublished data). We have designated the affected gene SEC36
, and the altered allele found in strain 271ts sec36-1
To test whether sec36-1
caused a general transport defect, we evaluated the transport of two other secretory proteins: Gas1p and invertase. Gas1p, a glycophosphatidylinositol-modified plasma membrane protein, acquires polysaccharide modifications in the Golgi, where it matures from a 105-kDa protein to a 125-kDa species. At the cell surface, Gas1p can be degraded by extracellular proteases (Sütterlin et al., 1997
), so we studied strains that contain the sec6-4
mutation, which prevents transport of proteins from the Golgi to the plasma membrane (Potenza et al., 1992
), to monitor Gas1p maturation without the complication of cell surface proteolysis. In pulse-chase experiments, the sec6-4
mutant exhibited significant processing of Gas1p to the 125-kDa species after 15 min at 38°C (Figure A). In contrast, the sec6-4 s36-1
double mutant strain exhibited no maturation of Gas1p in 15 min at 38°C (Figure A), indicating that sec36-1
blocks Gas1p maturation.
Figure 1 sec36-1 impairs the transport of Gas1p and invertase through the secretory pathway, and causes truncation of Sec36p. (A) sec6-4 (CKY151) and sec6-4 s36-1 (CKY728) cells were pulse labeled for 10 min with [35S]methionine and chased for (more ...)
The secretory protein invertase becomes hyperglycosylated during transport through the Golgi (Novick and Schekman, 1983
). In wild-type yeast, invertase matures to a heterogeneous array of 100–150-kDa bands. In a sec36-1
mutant, invertase migrated as a species smaller than 100 kDa, suggesting that it received less outer chain glycosylation that normal (Figure B). Furthermore, a significant portion of invertase was blocked in its secretion to the periplasm and accumulated intracellularly (Figure B). Collectively, these results indicate that the sec36-1
mutation causes a general block in secretory protein traffic at the restrictive temperature. However, invertase received more outer chain glycosylation in the sec36-1
mutant than in the ER-to-Golgi transport mutant sec12-4
(Figure B), suggesting that the block in transport occurred after invertase had passed through early Golgi compartments.
SEC36 Is in a Previously Uncharacterized ORF
To isolate the SEC36
gene, the pCT3 and YCp50 centromere-based yeast genomic DNA libraries (Rose et al., 1987
; Thompson et al., 1993
) were screened for clones that complement the inviability of the sec36-1
strain at 38°C. Five plasmids were identified that complemented both the CPY transport defects and temperature-sensitive growth defects. Each contained a 5-kb segment from the left arm of chromosome VII. Removal of a 700-bp Afl
II fragment internal to the YGL223c ORF on these library plasmids abolished their ability to complement the growth defects of a sec36-1
mutant (our unpublished data). Conversely, a plasmid containing only the YGL223c coding region expressed from the pGAL1
promoter (pRR35) restored growth to a sec36-1
strain at restrictive temperatures (our unpublished data).
To verify that YGL223c is at the sec36-1 locus, we integrated the URA3 marker by homologous recombination into chromosome VII, immediately adjacent to YGL223c. When this integrant was crossed to a sec36-1 mutant strain, the URA3 marker segregated with Ts+ in all 14 tetrads analyzed, showing that this region is tightly linked to the SEC36 locus.
SEC36 Encodes a Novel Protein That Is Truncated in sec36-1 Mutants
encodes an acidic 417 amino acid protein that is ~48 kDa. This was predicted by sequence analysis and confirmed by the demonstration that affinity-purified Sec36p antibody recognized a 50-kDa protein species in yeast lysates (Figure C). A nucleotide sequence was identified by the Génolevures project (http://cbi.labri.u-bordeaux.fr/Genolevures/Genolevures.php3
) that may code for a Sec36p homolog in the budding yeast species Saccharomyces exiguus
. Using the National Center for Biotechnology Information BLASTX algorithm, the translation product of this DNA fragment was shown to have 29% identity over a 231-amino acid region of Sec36p. No other sequences with significant similarities to the Sec36p amino acid sequence were found.
To define the mutation responsible for the defects in sec36-1
, we used the gap repair method (Orr-Weaver et al., 1983
), by which a segment of the SEC36
locus on a centromeric plasmid was excised, transformed into a sec36-1
mutant, and repaired by homologous recombination. The base sequence of the gap-repaired plasmids revealed a point mutation at position 594 (G to A), which converts the codon for Trp198 to a stop codon (Figure D). This mutation created a truncated gene product of about half the size of wild-type Sec36p that could be detected by immunoblots of lysates from sec36-1
mutant cells (Figure C).
Disruption of SEC36 Leads to a Severe Growth Defect
A disruption of the SEC36 locus was constructed by replacement of the YGL223c ORF in a wild-type diploid strain with kanMX6. Tetrads formed after sporulation of this diploid were dissected and grown at temperatures ranging from 18 to 37°C. The spore clones that carried that sec36::kanMX6 disruption formed only microcolonies at each temperature (our unpublished data). Identical results were obtained when a 700-base pair region after the first 11% of the YGL223c ORF was replaced by the TRP1 gene (our unpublished data). Viable spores containing sec36 disruptions could be recovered when wild-type SEC36 on a URA3-CEN plasmid is also present (our unpublished data). Unlike sister spores lacking the disruption, these cells were inviable on plates containing 5-fluoroorotic acid, showing their dependence on SEC36 for vigorous growth (our unpublished data). The observation that the C-terminal truncations caused by the sec36-1 mutation produced a much less severe growth defect than a complete gene deletion suggests that the N terminus of Sec36p is sufficient for its activity in the cell at most physiological temperatures.
Synthetic Lethality between sec36-1 and Mutations in COPI Coat Subunits and in Vesicle-docking and Fusion Proteins
To identify the stage of vesicle transport that is affected by sec36-1
, we crossed cells containing the sec36-1
mutation to strains with previously characterized secretory pathway blocks, and examined the meiotic progeny that resulted from sporulation of these diploids. It has been observed that mutations that impair the same stage of vesicle transport often display synthetic lethality, where the combinations of mutations are far more detrimental than the effect of each mutation on its own (Kaiser and Schekman, 1990
). As shown in Figure , the sec36-1
allele displayed only weak synthetic interactions with COPII vesicle formation mutations and Golgi-to-plasma membrane transport mutations. In contrast, it displayed severe synthetic growth defects with several vesicle docking and fusion mutations such as sec17-1
, as well as with all four of the COPI coat protein mutations tested. These data suggest that the function of SEC36
may be closely tied to both vesicle docking and fusion as well as COPI function.
Figure 2 sec36-1 is synthetically lethal with mutations that affect vesicle docking and fusion and COPI vesicle transport. A sec36-1 mutant (CKY726) was crossed to a set of mutants defining different steps in vesicle trafficking (Table ). Tetrads (more ...)
Sec36p Is in a Large Cytosolic Complex
To localize Sec36p, we used yeast cells containing Sec36p tagged at the C terminus with three HA epitopes as the only source of Sec36 protein. Spheroplasts from this strain were fractionated by differential centrifugation, and probed with monoclonal antibody to the HA epitope (12CA5). As shown in Figure A, Sec36p-HA was predominantly cytosolic. Similar results were obtained with wild-type extracts probed with Sec36p antibody (our unpublished data).
Figure 3 Sec36p-HA is in a large cytosolic complex. (A) Lysed spheroplasts derived from strain CKY729, expressing HA epitope-tagged Sec36p from a plasmid, were centrifuged at 13,000 and 100,000 × g. Pellet (P) and supernatant (S) samples for each fractionation (more ...)
To determine the size of native Sec36p, cytosolic samples from the strain containing Sec36p-HA were first precipitated with 40% ammonium sulfate and further purified over a DEAE-Sepharose anion exchange column. The pooled fractions containing Sec36p-HA were applied to a Superose 6 gel filtration column. Fractions from the Superose 6 column were analyzed by immunoblotting with HA antibody (12CA5). Fractions containing Sec36p-HA eluted from the Superose 6 column before the 670-kDa thyroglobulin marker (Figure B), placing Sec36p in a cytosolic complex with an estimated molecular mass of ~800 kDa. The same gel filtration profile was observed when samples from wild-type cells were analyzed with Sec36p antibody (our unpublished data).
Sec36p Coimmunoprecipitates with Sec34p-myc and Sec35p-myc
The yeast proteins Sec34p and Sec35p were recently found to reside in a large cytosolic complex involved in tethering ER-derived vesicles to the cis
-Golgi (Kim et al., 1999
; VanRheenen et al., 1999
). Because the sec36-1
mutant displayed characteristics of a vesicle-docking or fusion defect, we surmised that Sec36p might be in the Sec34p/Sec35p complex. Consistent with this hypothesis, we found that endogenous Sec36p efficiently coimmunoprecipitated with both Sec34p-myc
(Figure ). In the absence of tagged versions of either Sec34p or Sec35p, Sec36p was not immunoprecipitated, indicating that Sec36p formed a specific, direct or indirect, association with these proteins. The Sec36-1p protein, produced by a strain containing the sec36-1
mutation, did not efficiently interact with either Sec34p-myc
under these conditions (our unpublished data).
Figure 4 Sec36p coimmunoprecipitates with Sec34p-myc and Sec35p-myc. Wild-type cells (CKY10) were transformed with an empty vector (pRS305-2 μ), or with plasmids encoding Sec34p or Sec35p tagged at their C termini with c-myc epitopes (pRR65 and pRR66, (more ...)
Effects of sec36-1 and sec35-1 on Size of Complex Containing Sec34p, Sec35p, and Sec36p
As shown in Figure A, when yeast cell lysates were fractionated on a Superose 6 gel filtration column, Sec34p, Sec35p, and Sec36p exactly coeluted, consistent with all three proteins residing in the same complex. Based on these experiments, the apparent size of this complex is >800 kDa, which is larger than the size estimated from gel filtration analysis of lysates that were first precipitated with ammonium sulfate and fractionated by anion exchange chromatography (VanRheenen et al., 1999
; Figure B).
Figure 5 sec36-1 affects the mobility of the Sec34p/Sec35p complex. (A) Profiles of Sec34p, Sec35p, and Sec36p from wild-type (CKY10) cell extracts fractionated on a Superose 6 gel filtration column. Protein levels are expressed as a percentage of total protein (more ...)
To explore the relationship among Sec34p, Sec35p, and Sec36p further, we monitored the migration of these proteins through a Superose 6 gel filtration column during fractionation of extracts from cells containing either a sec36-1 or sec35-1 mutation. We found that when cytosol from a sec36-1 mutant was fractionated on the Superose 6 column, Sec34p and Sec35p still comigrated, but eluted later than from wild-type extracts, indicating that the size of the Sec34p/Sec35p complex had been altered by sec36-1 (Figure , B and C). Likewise, when whole-cell cytosol from a sec35-1 mutant was fractionated by Superose 6 chromatography, Sec36p eluted much later than from wild-type extracts (Figure ), suggesting that the complex in which Sec36p normally resides had been drastically altered by sec35-1. These results strongly suggest that Sec34p, Sec35p, and Sec36p are components of the same cytosolic complex. Moreover, they imply that disruption of this complex is the likely cause for the in vivo growth defects observed in strains containing either the sec35-1 or the sec36-1 mutation.
Figure 6 sec35-1 disrupts the Sec36p complex. (A) Profiles of Sec34p from wild-type (CKY10) and sec35-1 (GWY93) cell extracts fractionated on a Superose 6 gel filtration column. Protein levels are expressed as a percentage of total protein eluted from the column, (more ...)
Genetic Evidence for a Functional Link between Sec34p, Sec35p, and Sec36p
To evaluate whether Sec36p functions with Sec34p and Sec35p in cells, we analyzed the genetic relationships between SEC34
, and SEC36
. VanRheenen et al. (1999)
reported that the growth defects of strains containing sec34-1
mutations were suppressed by the SLY1-20
dominant allele on a low copy number plasmid. We found that this plasmid efficiently suppressed the growth defect of a sec36-1
mutant as well (Figure A), and partially suppressed the inviability of sec36::kanMX6
cells to the same extent that it partially suppressed the inviability of sec34
null cells (Figure B; VanRheenen et al., 1998
). Furthermore, as reported for overexpression of Sec34p, overexpression of Sec36p was found to partially suppress the sec35-1
mutation (Figure C; Kim et al., 1999
). However, overexpression of Sec34p or Sec35p did not affect the growth of sec36-1
strains (Figure A).
Figure 7 Genetic interactions between SEC36 and SLY1-20, SEC34, and SEC35. (A) A sec36-1 strain (CKY726) was transformed with low-copy plasmids containing SEC36 or SLY1-20 (pRR14 and pSV11, respectively), high-copy plasmids containing SEC34 or SEC35 (pSV25 and (more ...)
Loss of function alleles of SEC34
are each temperature sensitive for growth at 37°C, but sec34 s35
double mutants are inviable at 24°C (VanRheenen et al., 1999
). We found that sec36-1
was also synthetically lethal at 24°C with both the sec34-2
mutations (Figure D). A new allele of SEC34
, was obtained from another mutant recovered through the ribosome synthesis screen (strain 394ts; see MATERIALS AND METHODS). We found that sec36-1
was synthetically lethal with this novel allele as well (Figure D). Together, these data demonstrate that a close physical and functional relationship exists between Sec34p, Sec35p, and Sec36p.
Proteins Likely to be Components of Sec34p/Sec35p Complex Coimmunoprecipitate with Sec36p Antibody
Knowing that Sec36p is in the complex that contains Sec34p and Sec35p, we evaluated whether other components of this complex may associate with Sec36p as well. Accordingly, we found that affinity-purified Sec36p antibody immunoprecipitated a series of protein bands with apparent molecular masses of 35 kDa (p35), 51 kDa (p51), 70 kDa (p70), 100 kDa (p100), and 105 kDa (p105) (Figure A). These bands were not present in control immunoprecipitates with sec36-1 mutant extracts (Figure A), or immunoprecipitations with extracts from sec36::kanMX6 strains containing SLY1-20 (our unpublished data). Therefore, they likely represented Sec36p and proteins that specifically interact with full-length Sec36p.
Figure 8 Sec36p and Sec35p-myc coimmunoprecipitations. (A) Proteins that immunoprecipitate with Sec36p. Wild-type (CKY10) and sec36-1 mutant (CKY726) cells were labeled with [35S]methionine for 3 h before lysis and immunoprecipitation with affinity-purified (more ...)
After Sec36p antibody immunoprecipitates from wild-type cells were washed with 0.2% SDS or 2 M urea, only p51 remained tightly associated with the antibody (Figure A), indicating that this species was Sec36p itself, consistent with the predicted size for Sec36p of 48 kDa. A 30-kDa band (p30) was observed in immunoprecipitations from a sec36-1 mutant that was not present in immunoprecipitations from wild-type lysates (Figure A). This band probably corresponds to the truncated protein produced by the sec36-1 mutation.
The remaining p105, p100, p70, and p35 protein bands were likely to be proteins that associate with Sec36p. Because Sec34p-myc
both efficiently immunoprecipitated Sec36p, it was reasonable that Sec36p antibody coimmunoprecipitated Sec34p and Sec35p. Sec34p migrates at 105 kDa by SDS-PAGE (Kim et al., 1999
), so it was likely that the p105 band was Sec34p. The predicted molecular mass of Sec35p is ~32 kDa, so the p35 band was likely to be Sec35p. It was unlikely that the p70 and p100 bands were fragments of Sec34p because significant degradation of endogenous Sec34p had not been observed by immunoblotting with Sec34p polyclonal antibody (our unpublished data). Therefore, these species probably represented previously uncharacterized components of the Sec34p/Sec35p/s36p complex. Initial efforts to obtain enough material to identify these additional proteins by mass spectrometry from complexes isolated by affinity chromatography on a Sec36p antibody column, or using Sec36p tagged with glutathione S
-transferase, were unsuccessful (our unpublished data).
Proteins Encoded by ORFs YNL041c and YPR105c Coimmunoprecipitate with Sec35p-myc
To identify additional components of the Sec34p/Sec35p/s36p complex, we next performed large-scale c-myc antibody (9E10) immunoprecipitations with extracts from cells overexpressing Sec35p-myc. In these experiments, several protein bands (p70, p90, p95, and p100) seemed to specifically coimmunoprecipitate with Sec35p-myc, indicated as p45 (Figure B). Other Sec35p-myc–interacting proteins may have been missed in our analysis because we used strains expressing abnormally high levels of Sec35p-myc. For example, endogenous Sec36p was also coimmunoprecipitated with Sec35p-myc in these experiments, as detected by immunoblotting with Sec36p antibody (our unpublished data), but a significant Coomassie-stained protein band around the size of endogenous Sec36p was not observed in elution fractions (Figure B).
All four of the protein bands that specifically coimmunoprecipitated in our experiments were digested with trypsin, and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The set of peptides in which mass-to-charge (m/z) values were >1000 were used to identify the proteins contained in these samples (Table ). At a mass tolerance of ±25 parts per million (ppm), 10/35 peptides from the p100 band matched the YPR105c-encoded protein, and a nonoverlapping set of 15/35 peptides from the p100 band matched the YNL041c-encoded protein. No matches were found for the p90 and p95 fragments even when the mass tolerance was increased to ±50 ppm, so the proteins contained in these two band remain unidentified. At a mass tolerance of ±50 ppm, 11/20 peptides from the p70 band matched the YPR105c-encoded protein. To demonstrate the significance of this result, the region corresponding to the p70 fragment from the immunoprecipitation lacking Sec35p-myc was also analyzed by mass spectrometry. It yielded no matches, even with a higher mass tolerance window.
Mass spectrometry analysis of proteins that coimmunoprecipitate with Sec35p-myc
Based on their predicted amino acid sequences, the YNL041c- and YPR105c-encoded proteins are estimated to be 97 and 99 kDa, respectively (Saccharomyces Genome Database, Stanford University, Stanford, CA). It was, therefore, likely that the 100-kDa band contained a mixture of these proteins, and that the 70-kDa band was a degradation product of the YPR105c-encoded protein.
Importantly, results from recent large-scale yeast two-hybrid screens show that the YNL041c-encoded protein interacts with both Sec35p and Sec36p, and that the YPR105c-encoded protein interacts with Sec35p (Uetz et al., 2000
; Ito et al., 2001
). These interactions strongly suggest that both YNL041c and YPR105c encode proteins that physically associate with Sec34p, Sec35p, and Sec36p. Consequently, we have designated YNL041c and YPR105c as SEC37
SEC37 and SEC38 Encode Conserved Proteins
encode proteins that are probably cytosolic or peripherally associated with membranes, because their predicted sequences lacked obvious transmembrane domains. Sec37p and Sec38p share significant amino acid sequence similarity along their lengths with predicted proteins from other budding yeast species (http://cbi.labri.u-bordeaux.fr/Genolevures/Genolevures.php3
) as well as from other eukaryotes (Table ). Therefore, Sec37p and Sec38p seem to be highly conserved eukaryotic proteins. For Sec38p, the Arabidopsis thaliana
homolog is distinguished from the others by the presence of an N-terminal extension (Table ). For Sec37p, the S. cerevisiae
protein is longer than related proteins from other species, primarily due to a 100–200-amino acid extension at the N terminus (Table ).
Disruption of SEC37 Produces Defects Similar to Those Caused by Mutations in SEC34 and SEC35
We found that haploid cells in which the SEC37 was replaced with kanMX4 grew on complete medium at temperatures ranging from 24 to 37°C (our unpublished data). Therefore, SEC37 was apparently dispensable for robust growth at physiological temperatures.
To test whether sec37::kanMX4
cells have any defect in vesicle-mediated transport, we monitored the transport of CPY through the secretory pathway by pulse-labeling and immunoprecipitation. In wild-type cells, CPY was efficiently processed to its mature form after 1 min, whereas in the sec37::kanMX4
strain, CPY was delayed in its conversion from the ER-modified p1 form to the Golgi-modified p2 form (Figure A). The p1 form of CPY was previously shown to accumulate in ER-to-Golgi secretory pathway mutants, including strains containing mutations in SEC34
(Li and Warner, 1996
; Wuestehube et al., 1996
Figure 9 Disruption of SEC37 slows the rate of ER-to-Golgi transport and is synthetically lethal with sec34-2 and sec35-1 at 24°C. (A) Wild-type (CKY10) cells, sec36-1 (CKY726) cells, and sec37::kanMX4 (CKY733) cells were pulse labeled with [35 (more ...)
For evidence that disruption of SEC37 specifically affected the complex that contains Sec34p and Sec35p, a sec37::kanMX4 halpoid strain was crossed to strains containing sec34-2, sec35-1, and sec36-1 alleles. As shown in Figure B, deletion of SEC37 was synthetically lethal at 24°C with sec34-2 and sec35-1, but not with sec36-1.
SEC38 Is Essential and Displays Genetic Interactions Consistent with a Functional Link to SEC34 and SEC35
Haploid cells in which SEC38
was replaced by kanMX4
were inviable at 24°C, and could not form visible microcolonies (Figure A). However, in the presence of the SLY1-20
plasmid, these sec38::kanMX4
cells formed visible microcolonies (Figure A). Previous results have implied that the ability to be suppressed by SLY1-20
is a hallmark of mutations in genes that encode proteins with functions related to Sec34p and Sec35p function (VanRheenen et al., 1998
; Figure ). Therefore, this result suggests that the function of Sec38p is related to that of Sec34p and Sec35p. Likewise, it was observed previously that overexpression of Sec34p, Sec35p, or Sec36p, suppressed the temperature-dependent growth defects of a sec35-1
mutant strain at 33°C, and we found that overexpression of Sec38p has a similar effect (VanRheenen et al., 1998
; Figures C and B). Collectively, these results support a functional as well as physical connection between Sec38p and Sec34p, Sec35p, and Sec36p. Our results are consistent with those published elsewhere during submission of this article, in which Sec38p/Sgf1p was isolated as a high-copy suppressor of the temperature sensitivity of sec35-1
that interacts with Sec35p and cofractionates with Sec34p and Sec35p (Kim et al., 2001
Figure 10 Genetic interactions between the essential SEC38 gene and both SLY1-20 and SEC35. (A) Diploid strain in which one copy of the SEC38 locus was disrupted with kanMX4 (CKY736) was transformed with a URA3-marked plasmid containing SLY1-20 (pSV11). The untransformed (more ...)
Disruption of SEC37 or SEC38 Affects Size of Complex Containing Sec34p and Sec35p
To determine how Sec37p and Sec38p might influence the complex that contains Sec34p and Sec35p, extracts from cells containing either SEC37 disrupted with kanMX4 or SEC38 disrupted with kanMX4 were fractionated on a Superose 6 gel filtration column. When sec37::kanMX4 cell extracts were fractionated this way, Sec34p, Sec35p, and Sec36p still comigrated through the column, but were slightly delayed relative to their migration during fractionation of wild-type extracts (Figure ), implying that deletion of Sec37p results in a partial disruption of the complex (Table ).
Figure 11 Deletion of SEC37 affects the mobility of the Sec34p/s35p complex. (A) Profiles of Sec34p from wild-type (CKY10) and sec37::kanMX4 (CKY733) cell extracts fractionated on a Superose 6 gel filtration column. Protein levels are expressed as a percentage (more ...)
Characteristics of components of the yeast Sec34p/Sec35p complex
Because SEC38 is essential, to analyze the effects of deleting SEC38, we used a sec38::kanMX4 strain containing the SLY1-20 plasmid. We found that the SLY1-20 plasmid did not affect the elution of Sec34p or Sec35p from the Superose 6 column (Figure A). Disruption of SEC38, however, had a dramatic effect on the migration of Sec34p and Sec35p from the column (Figure B). These two proteins no longer coeluted and were largely distributed to distinct fractions. This result suggests that Sec38p may be necessary for Sec34p and Sec35p to interact, as well as for the integrity of the complex as a whole. This may be why disruption of SEC38 had such a drastic effect on cell viability. Conversely, the more subtle effects that loss of SEC37 has on the complex may explain why disrupting that gene did not cause a significant growth defect.
Figure 12 Deletion of SEC38 disrupts the Sec34p/Sec35p complex. (A) As a control to show that the presence of SLY1-20 does not significantly alter the mobility of the complex, Sec34p and Sec35p were fractionated on a Superose 6 gel filtration column from wild-type (more ...)
Evidence That Cod4p, Cod5p, and Dor1p Function in Sec34p/Sec35p Complex In Vivo
While this article was in preparation, Whyte and Munro (2001)
reported isolation of a complex containing the proteins Cod4p, Cod5p, and Dor1p in addition to the five subunits of Sec34p/Sec35p complex that we have described herein. We were interested, therefore, to extend our genetic and biochemical analyses to these three additional gene products. We first tested disruption of COD4
, and DOR1
for their effects on cell viability and protein transport. Although strains containing cod4::kanMX4
, and dor1::kanMX4
alleles display wild-type growth at a range of temperatures, we found that each of these mutations causes a severe synthetic growth defect in combination with the sec35-1
allele at 24°C (Figure A). A strain carrying dor1::kanMX4
displayed the most severe synthetic growth defect, and also displayed a significant delay in the maturation of CPY from p1 (ER form) to p2 (Golgi form) (Figure B).
Figure 13 Tests for the involvement of Cod4p, Cod5p, and Dor1p in the Sec34p/Sec35p complex. (A) A sec35-1 mutant (GWY93) was crossed to cod5::kanMX4 (Y04373), dor1::kanMX4 (Y06484), and cod4::kanMX4 (Y07209) strains. Tetrads from the resulting diploids were incubated (more ...)
To test the effect of these alterations on the complex that contains the majority of Sec34p and Sec35p, we prepared cytosol from each disruption strain and determined the size of the Sec34p/Sec35p complex by gel filtration. All three deletions caused a slight shift in the mobility of Sec34p and Sec35p that was similar to that observed when SEC37 was deleted (compare Figure C with Figure ).