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. 2001 Apr;12(4):1035-45.
doi: 10.1091/mbc.12.4.1035.

The ADP ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have overlapping, but not redundant functions in retrograde transport from the Golgi to the endoplasmic reticulum

A Spang  1 , J M Herrmann, S Hamamoto, R Schekman
Affiliations
Free PMC article

The ADP ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have overlapping, but not redundant functions in retrograde transport from the Golgi to the endoplasmic reticulum

A Spang et al. Mol Biol Cell. 2001 Apr.
Free PMC article

Abstract

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the gamma-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Deltagea1 Deltaarf1 mutant is not more sickly than a Deltaarf1 strain, whereas Deltagea2 Deltaarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

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Figures

Figure 1
Figure 1
GEA2 is a multicopy suppressor of sec21-3. Strains were grown overnight at the permissive temperature in synthetic complete medium-URA to select for the plasmids. Cells were diluted to a final concentration of 106 cells/ml, and 10 μl of 10-fold dilutions were spotted on YPD plates. The plates were incubated at the indicated temperatures. GEA2 overexpression from a 2μ plasmid restored viability of sec21-3 at 35°C. wt, wild type.
Figure 2
Figure 2
GEA1 and GEA2 genetically interact with coatomer. Mutant strains were transformed with a 2μ plasmid (pRS426) either empty or bearing GEA1 or GEA2. Strains were grown and treated as described in Figure 1. The temperatures indicated were chosen because considerable growth of the mutant strains with the empty vector was observed. (A) GEA1 and GEA2 do not suppress sec21-1 and sec21-2. GEA2 suppressed specifically the sec21-3 allele and did not significantly alter the growth of sec21-1 and sec21-2. In contrast, GEA1 overexpression enhanced the ts phenotype of the sec21-2 mutant, whereas sec21-1 was unaffected. (B) GEA1 genetically interacts with sec33-1 and sec27-1. Similar to the effect observed in the sec21-2 mutant, GEA1 overexpression enhanced the ts sensitivity. In contrast, multicopy GEA1 suppressed sec27-1 at 33°C.
Figure 3
Figure 3
GEA1 GEA2 conditional double mutants accumulate membranes at the nonpermissive temperature. gea1-4 Δgea2 cells were grown to early log phase in YPD at 23°C (permissive temperature). The culture was split in half. One-half remained at 23°C, and the other half was transferred to 37°C (restrictive temperature) for 1 h. The cells were prepared for electron microscopy and ultrathin sections were stained. (A and B) Cells grown at 23°C. (C and D) Cells that were shifted to 37°C for 1 h. Note the membrane accumulation, especially ER membranes at 37°C and Golgi structures even at 23°C. Some examples are highlighted with arrows. Pictures were taken at the same magnification. Bar, 0.9 μm.
Figure 4
Figure 4
ARF-GEF requirement for retrograde transport from the Golgi to the ER. Membranes of mutant and wild-type strains were prepared from cells grown at a permissive temperature. The membranes were added to stage III of the retrieval assay (for details see MATERIALS AND METHODS). After incubation at 20°C, the samples were complemented with coatomer and Arf1p. Retrieval reactions were incubated at 30°C. At the end of the reaction, samples were subjected to trypsin digestion, concanavalin A-Sepharose precipitation, and SDS-PAGE. The species migrating at the position of trimmed gpαF represents the reporter that has been transported back to the ER. Representative gels are shown. (A) Gea1p and Gea2p support retrograde transport. (B). Sec7p is not required for retrieval of reporter molecules back to the ER. Two different ts mutants were compared with wild-type membranes. In the control, the stage IV (retrieval from the Golgi to the ER) was performed at 4°C.
Figure 5
Figure 5
Gea1p and Gea2p are required for the formation of retrograde transport vesicles. Golgi-enriched membranes from either wild-type or gea1-4 gea2:: HIS3 cells were prepared from cells grown at a permissive temperature. A Golgi budding assay was performed in the presence of Arf1p, guanosine 5′-[γ-thio]triphosphate, and coatomer at 30°C. The reaction sample was loaded on a Ficoll/sucrose gradient and centrifuged for 2 h at 100,000 × g. Fractions were collected from the top and concentrated by trichloroacetic acid precipitation, and proteins were separated by SDS-PAGE and analyzed by immunoblot with antibodies directed against Sec21p, Emp47p, Bos1p, and Sec22p. The arrows indicate the direction of the gradient from the top to the bottom. Golgi membranes sedimented to the bottom of the gradient and soluble proteins remained at the top. Coated vesicles peaked in fractions 6 and 7.
Figure 6
Figure 6
Gea1p and Gea2p are peripheral membrane proteins. (A) Strains that contained either a chromosomal C-terminal myc-tagged Gea1p or Gea2p were grown in YPD to early to mid log phase. Cells were harvested, gently lysed, and subjected to differential centrifugation. To enrich for Gea1p and Gea2p, we subjected the supernatant (S) and the solubilized pellets (P) to immunoprecipitation with anti-myc antibodies coupled to proteinA-Sepharose. The precipitates were analyzed by immunoblot decorated with anti-myc-antibodies. (B) Gea2p is peripherally associated with membranes. YAS61, which contains a chromosomal Gea2p-myc-his6, was grown to early log phase. Cells were harvested and lysed with glass beads under native conditions. Aliquots of the lysate were supplemented with either 1 M NaCl or 0.2% Triton X-100 (TX-100) before differential centrifugation. Samples were analyzed by immunoblot with antibodies directed against the myc-epitope, Anp1p and Pgk1p.
Figure 7
Figure 7
Gea2p localizes to the Golgi apparatus. Cells that bear chromosomal tagged forms of GEA2, EMP47, and OCH1 were grown to early log phase and prepared for immunofluorescence. The proteins were visualized by incubation with a monoclonal anti-myc (Gea2p-myc-his6 and Emp47p-myc) or anti-HA (Och1p-HA) antibody followed by a secondary anti-mouse antibody coupled to CY3. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI).
Figure 8
Figure 8
Gea1p and Gea2p show different localization patterns. Strains YAS61 (Gea2p-myc-his6) and YAS88 (Gea1p-myc) were grown in YPD to early to mid log phase and prepared for immunofluorescence. The GEFs were stained with anti-myc antibodies followed by anti-mouse coupled to CY3 antibodies. The DNA was visualized with 4′,6-diamidino-2-phenylindole.
Figure 9
Figure 9
Gea1p and Gea2p cofractionate with cis-Golgi proteins. Strain YAS61 (Gea2p-myc-his6) was grown to early log phase. A membrane pellet of a 10,000 × g spin was floated to equilibrium on a sucrose gradient. Fractions were collected from the top. The fractions were either separated by SDS-PAGE, and analyzed by immunoblot with antibodies directed against Gea1p, the myc-epitope (Gea2p-myc-his6), Anp1p, Emp47p, Sed5p, Bos1p, and Arf1p (A) or sampled for GDPase activity assays (B). The arrow in A indicates the direction of movement of membranes within the gradient.
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