FIGURE 1:

Roy1 interacts with Ypt52 under physiological conditions. (A) The interaction of the indicated small GTPases with Roy1 was determined with a two-hybrid assay by the expression of the HIS3 gene or the LacZ reporter gene. (B) The indicated yeast cells were lysed using glass beads and a multibead shocker. Lysates were subjected to IP with anti-Ypt52 antibodies, and the resulting precipitates and total cell lysates were subjected to IB analysis with indicated antibodies. (C) The indicated recombinant proteins purified from Sf21 insect cell lysates or E. coli BL21 (DE3) cell lysates were analyzed by SDS–PAGE and visualized with Coomassie staining. The positions of recombinant proteins are indicated. (D) The indicated recombinant proteins were mixed and incubated at 30°C for 30 min. Binding of Ypt52 and the Roy1–Skp1 complex was detected by GST pull-down analyses. (E) The indicated cells were cultured to an OD₆₀₀ = 0.6 and then incubated in the presence of 0.25% DMSO or 50 μM MG132 for 2 h. Cells were harvested and lysed by the TCA lysis method. Extracts were subjected to IB with indicated antibodies. (F and G) The indicated cells were cultured to an OD₆₀₀ = 0.8 and then incubated in the presence of 200 μg/ml cycloheximide. Cells were harvested and lysed by the TCA lysis method at the indicated time intervals. Extracts were subjected to IB with the indicated antibodies. (H) The indicated lysates were prepared as described in B and subjected to IP with anti-FLAG antibodies. The resulting precipitates and total cell lysates were subjected to IB with indicated antibodies. NS: nonspecific band.