Figure 5.

Rot1 is a molecular chaperone for Big1. (A) CHX chase of Big1-HA. ROT1 and rot1-2 cells expressing Big1-HA (YM84 and YM85) were grown in YPD at 23°C and incubated at 37°C for 2 h. The cells were further incubated with CHX (0.1 mg/ml) for indicated period at 37°C. The cell lysates were subjected to Western blotting to detect Big1-HA. (B) Big1-HA is degraded by the ERAD in the rot1-2 cells. CHX chase experiments were performed as in A using the BIG1-HA strains: YM84, YM85, and YM86 (ROT1 ubc7Δ); YM87 (rot1-2 ubc7Δ); YM96 (ROT1 kar2-1); and YM97 (rot1-2 kar2-1). The signal intensity of Big1-HA was quantified, and the averages and SDs of at least four independent experiments are shown. (C and D) Rot1 interacts with Big1-HA. Lysis of YM84 and YM85 cells and IP were performed, and results are presented as Figure 4, C and D.