Figure 7.

Pma1p-transporting SVs contain Drs2p. (A) Immunoblots of Nycodenz gradient fractions from 38°C-shifted and 27°C-grown sec6-4 cells (same gradients as shown in Figure 1) were probed with antibodies against HA-tagged Pma1p, Drs2p, and the ER membrane protein Dpm1p. (B) Protein G-coupled Dyna beads loaded with anti-HA antibody were used to immuno-isolate Pma1p-HA–containing membranes from plasma membrane-ATPase peak fractions (6–8) of the 38°C-shifted sec6-4 Nycodenz gradient. In one reaction, the beads were preincubated with a peptide corresponding to the HA epitope to control for the specificity of the immunoisolation procedure (+HA peptide). Membranes bound to the beads (B) or collected from the supernatant (S) were analyzed by immunoblotting using antibodies against HA-tagged Pma1p and Drs2p.