Figure 9.
Membrane association of wild-type and mutant Vps35 proteins. Spheroplasts from a vps35Δ strain (PBY1) carrying plasmids encoding either the wild-type (pLS13), vps35–101 (pLS12), vps35–103 (pPB7–9.7), vps35–104 (pPB7–10.1), vps35–107 (pPB7–108.5), or vps35–108 (pPB7–010.4) alleles were pulsed for 45 min with [35S]methionine/cysteine and chased for 15 min with unlabeled amino acids. The spheroplasts were then lysed, and lysates were centrifuged at 13,000 × g to generate a pellet fraction (P13). The supernatant was then centrifuged at 150,000 × g to generate pellet (P150) and supernatant (S150) fractions. Wild-type and mutant Vps35p were immunoprecipitated from each fraction and analyzed by SDS-PAGE and fluorography. The relative percentage of Vps35p present in each fraction as determined by Phosphorimager analysis is indicated below each panel.