Figure 6

ERV14 promotes capture of Yor1 into COPII vesicles. (A) The genetic interaction profile of the erv14-Δ0 strain clustered with those for the sys1-Δ0, arl3-Δ0, mak3-Δ0, and mak10-Δ0 strains. Columns represent diverse gene-drug interactions as described in methods. (B) Based on known data [55], and supported by our phenotypic and molecular findings, Erv14, Sys1, Arl3, Mak3, and Mak10 appear to function in a pathway with Erv14 acting in the ER and Sys1, Arl3, Mak3, and Mak10 functioning in the Golgi. (C) Gene-oligomycin interactions for erv14-Δ0 strains, in the context of either wild-type Yor1 or Yor1-ΔF, suggested Erv14 promotes the biogenesis of both Yor1 and Yor1-ΔF. (D) Packaging of wild-type Yor1 into COPII vesicles was quantified using an in-vitro budding assay that measures capture of newly synthesized cargo proteins from radio-labeled permeabilized cells after addition of purified COPII proteins in the presence ('+') or absence ('-') of GTP [9], followed by immunoprecipitation of the cargo protein of interest. 'T' indicates the total membrane pool of labeled Yor1-HA. Erv14-containing membranes showed approximately four-fold more efficient capture into vesicles of HA-tagged Yor1 than erv14-Δ0 membranes. The defect showed specificity, since ERV14 deletion did not affect packaging of another cargo protein, Sec22. Quantification of three independent experiments is shown at right; error bars represent standard deviation. (E) Similar vesicle budding assays from mak10-Δ0, sys1-Δ0, and arl3-Δ0 membranes showed no defects in Yor1 capture into COPII vesicles in these mutants, suggesting they function downstream of Erv14.