Figure 7. Sec17 overproduction partially rescues in vivo activity of Qc-5∆.

(A) Analysis of cis-SNARE complex abundance in lysates of cells overproducing Sec17 and Sec18. The top panel shows immunoblots of cell lysates. In the bottom panel, anti-Vam3 (Qa-SNARE) was immunoprecipitated from detergent lysates from the indicated strains under non-denaturing conditions. The precipitated material was separated by SDS-PAGE and analyzed by immunoblot, as indicated. IP, immunoprecipitate; FT, flow-through. PGK, phosphoglycerate kinase (control). Additional experimental details are provided in the Materials and methods. (B) Vacuoles in the indicated cell lines were labeled by pulse-chase with FM4-64 dye and observed by epifluorescence. (C) Quantification of phenotypes in B. Bars show mean scores from three independent experiments (n = 88–354 cells per genotype per experiment). (D) Vacuoles in the indicated cell lines were labeled by pulse-chase with FM4-64 dye and observed by epifluorescence. (E) Quantification of phenotypes in D. Bars show mean scores as in C.

Figure 7—figure supplement 1. Little or no rescue of fragmented vacuole morphology was observed with Qc-3∆ or Qc-5∆ chromosomal integrants when Sec17, or Sec17 and Sec18, were overproduced in a YCK3 genetic background.

Vacuoles were labeled by pulse-chase with FM4-64 dye and observed by epifluorescence. Representative fields are shown.

Figure 7—figure supplement 2. Ability of SEC17 mutants to support growth in sec17-1 mutant cells at nonpermissive temperature.

Liquid cultures were adjusted to equivalent cell densities, serial dilutions were spotted onto synthetic complete agar plates lacking uracil to select for plasmid retention. The plates were incubated at permissive (25°C) or nonpermissive (37°C) temperature for growth of sec17-1 cells, and photographed.