PMC

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, as detected by immunoblotting. (B) Profiles of Sec36p from wild-type (CKY10) and sec35-1 (GWY93) cell extracts fractionated on a Superose 6 column.

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 of total protein eluted from the column, as detected by immunoblotting. (B) Profiles of Sec35p from wild-type (CKY10) and sec37::kanMX4 (CKY733) cell extracts fractionated on a Superose 6 column. (C) Profiles of Sec36p from wild-type (CKY10) and sec37::kanMX4 (CKY733) cell extracts fractionated on a Superose 6 column.

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, respectively). Extracts from these transformants were subjected to c-myc antibody (9E10) immunoprecipitations. The presence of Sec36p in total cell extracts and in immunoprecipitates was monitored by SDS-PAGE followed by immunoblotting with affinity-purified Sec36p antibody (right). The presence of tagged Sec34p or Sec35p in total cell extracts was monitored by SDS-PAGE and immunoblotting with c-myc antibody (9E10; left). Relative amounts of material loaded per lane are noted below the images.

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 eluted from the column, as detected by immunoblotting. The elutions of the 670-kDa thyroglobulin, 200-kDa β-amylase, and 67-kDa bovine serum albumin size standards from the Superose 6 column, and the void volume, are indicated. (B) Profiles of Sec34p from wild-type (CKY10) and sec36-1 (CKY726) cell extracts fractionated on a Superose 6 column. (C) Profiles of Sec35p from wild-type (CKY10) and sec36-1 (CKY726) cell extracts fractionated on a Superose 6 column.

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 were incubated for several days on rich medium at a range of temperatures (24–38°C). The height of each bar indicates the restrictive temperature for the mutation. The shaded region indicates restrictive temperatures for the corresponding double mutant with sec36-1. All double mutants with restrictive temperatures of 24°C formed microcolonies on the tetrad dissection plates.

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 (CKY10) cell extracts containing SLY1-20 (pSV11). An asterisk (∗) designates the peak of Sec34p and Sec35p elution during fractionation of extracts from wild-type (CKY10) cells lacking SLY1-20. Protein levels are expressed as a percentage of total protein eluted from the column, as detected by immunoblotting. (B) Profiles of Sec34p and Sec35p from extracts of sec38::kanMX4 cells containing SLY1-20 (CKY735) fractionated on a Superose 6 column.

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 (left) and transformed (right) diploids were sporulated at 24°C on rich medium. Some of the microcolonies that arose are marked by arrows. Tetrad analysis revealed that all inviable spores and microcolonies contained sec38::kanMX4. (B) sec35-1 mutant cells (GWY93) were transformed with a low-copy plasmid containing SLY1-20 (pSV11), a high-copy plasmid containing SEC38 (pRR72), or a vector plasmid (pRS426). Serial 10-fold dilutions were spotted onto rich medium at 30 or 33°C and grown for several days.

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 [³⁵S]methionine for 10 min at 37°C, followed by a chase for the indicated times, and immunoprecipitation with CPY antibody to monitor the conversion of CPY from the p1 (ER-modified) form, to the p2 (Golgi-modified) and mature (vacuole processed) forms. (B) sec37::kanMX4 (CKY733 or CKY734) cells were crossed to sec34-2 (GWY95), sec35-1 (GWY93), and sec36-1 (CKY726) cells. After sporulation, tetrads were dissected and incubated at 24°C on rich medium. Spores that seemed inviable actually formed microcolonies that were inferred to be double mutants by tetrad analysis.

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 pSV17, respectively), or vector alone (pRS306-2 μ), and grown on rich medium at 24 or 37°C. (B) Diploids in which one copy of SEC36 was disrupted by kanMX6 (CKY730) were transformed with a plasmid containing SLY1-20 (pSV11) and sporulated. Tetrads from untransformed (left) and transformed (right) cells were dissected and incubated on rich medium at 24°C. Arrows indicate examples of colonies of sec36::kanMX6 cells containing SLY1-20. (C) A sec35-1 mutant (GWY93) was transformed with a plasmid containing SLY1-20 (pSV11), a plasmid that overexpresses Sec36p (pRR31), or vector alone (pRS306-2 μ). Serial 10-fold dilutions were spotted onto rich medium and incubated at 30 or 33°C. (D) A sec36-1 mutant (CKY726 or CKY727) was crossed to a sec34-2 mutant (GWY95), sec34-3 mutant (CKY731), and a sec35-1 mutant (GWY93). Tetrads from the resulting diploids were incubated for several days at 24°C on rich medium.

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 15 min at 38°C. Radiolabeled Gas1p was detected by immunoprecipitation with Gas1p antibody, followed by SDS-PAGE and PhosphorImager analysis. (B) Wild-type (CKY10), sec36-1 (CKY726), and sec12-4 (CKY39) cells were transformed with pNV31, which encodes invertase expressed from the constitutive TPI1 promoter. Transformants were pulse labeled for 10 min with [³⁵S]methionine and chased for 15 min at 37°C before they were converted into spheroplasts. After centrifugation at 500 × g, pools of intracellular (I) and secreted (S) invertase were detected from pellet and supernatant fractions, respectively, by immunoprecipitation with invertase antibody. (C) Protein extracts from wild-type (CKY10) and sec36-1 (CKY726) cells were immunoblotted with affinity-purified Sec36p antibody. To detect the Sec36-1p protein, the blot was overexposed, revealing an immunoreactive band unrelated to Sec36p, indicated by an asterisk (∗). (D) The sec36-1 mutation converts codon 198 to a stop codon.

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 step were analyzed by SDS-PAGE and immunoblotting with either HA antibody (12CA5) to detect Sec36p-HA or antibody to the membrane marker protein Pma1p. (B) CKY729 cells were lysed with glass beads and centrifuged at 100,000 × g. The supernatant was precipitated with 40% ammonium sulfate, fractionated on a DEAE-Sepharose anion exchange column, and then applied to a Superose 6 gel filtration column. The Superose 6 column fractions were analyzed by immunoblotting with HA antibody (12CA5) and the fractions that contain all detectable Sec36p-HA are shown. Fraction numbers and the migration of the thyroglobulin size standard (670 kDa) are indicated.

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 Sec36p antibody. Two of the samples from wild-type cells were washed with buffer containing either 0.2% SDS or 2 M urea before SDS-PAGE, as indicated. Arrows indicate Sec36p, Sec36–1p, and other proteins that specifically coprecipitated with Sec36p. Asterisks (∗) mark two bands that seem to be specific to the Sec36p immunoprecipitation in this experiment, but varied in intensity between experiments. The migration of molecular weight markers is indicated on the right. (B) Proteins that immunoprecipitate with Sec35p-myc. Lysates from wild-type (CKY10) cells transformed with a plasmid expressing Sec35p-myc (pRR66) or a vector control (pRS305–2 μ) were centrifuged at 100,000 × g. The supernatant was incubated with c-myc antibody (9E10) conjugated to Sepharose beads. Samples were washed extensively and eluted with 100 mM glycine, pH 2.5. Eluted material was TCA precipitated and resolved by SDS-PAGE. Coommassie-stained bands that were specifically immunoprecipitated when Sec35p-myc was present are indicated with arrows. The migration of molecular weight markers is noted on the left.

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 for several days at 24°C on rich medium. Microcolonies and inviable spores were inferred to be double mutants based on the phenotypes of the remaining spores. (B) Wild-type (CKY10) cells, and cod5::kanMX4 (Y04373), dor1::kanMX4 (Y06484), and cod4::kanMX4 (Y07209) cells containing a plasmid with the MET15 gene to complement their methionine auxotrophy were pulse labeled with [³⁵S]methionine for 10 min at 37°C, followed by a chase for the indicated times. Cell extracts were immunoprecipitated with CPY antibody to monitor maturation of CPY. (C) Extracts from cod5::kanMX4 (Y04373), dor1::kanMX4 (Y06484), or cod4::kanMX4 (Y07209) cells were fractionated on a Superose 6 column and probed with Sec34p antibody. Identical profiles were seen when fractions were probed with Sec35p antibody. Protein levels are expressed as a percentage of total protein eluted from the column, as detected by immunoblotting. An asterisk (∗) designates the peak of Sec34p and Sec35p elution during fractionation of extracts from wild-type (CKY10) cells.