Figure 10.

The trs120-2, -4, and -8 mutants display a defect in the localization of coatomer subunits. (A) Wild-type (wt) and mutant cells containing Sec21p-GFP, Sec7p-GFP, or Och1p-HA were grown to early log phase at 25°C and then shifted to 37°C for 15 min. For the analysis of Och1p-HA, cells were processed for immunofluorescence using a monoclonal anti-HA antibody. (B) Wild-type and mutant cells containing Ret2p-GFP were grown at 25°C and then shifted to 37°C for 15 min. (C) To immobilize TRAPP on beads, 50 mg of lysate prepared from a Bet3p PrA-tagged strain (lanes 2 and 3) was incubated with 30 μl of IgG–Sepharose beads for 2 h at 4°C. As a control, an untagged lysate (lane 1) was incubated with beads in the same way. The beads were washed three times with 1 ml of wash buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl₂, 1% Triton X-100, and 1× Protease inhibitor cocktail) and then incubated with 50 mg of wild type (lanes 1 and 2) or ret1-1 lysate (lane 3) for 3 h. After the incubation, the beads were washed four times with 1 ml of wash buffer, eluted with 0.2 M glycine, pH 2.8, and neutralized before loading onto an SDS–polyacrylamide gel. To detect Ret1p in lysates, equal amounts of wild-type (lane 4) and ret1-1 (lane 5) lysates were subjected to SDS gel electrophoresis and blotted with anti-coatomer antibody. (D) Lysates prepared from a Ret1p TAP-tagged (lane 2) or untagged (lane 1) strain were incubated with 30 μl of IgG–Sepharose beads for 2–3 h at 4°C. The beads were washed and treated as described in C. The lysate containing TAP-tagged Ret1p (lane 3) was subjected to SDS-PAGE and blotted with anti-Trs33p and anti-Sec13p antibodies.