Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D. (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.