Figure 6.
The relationship of Rot1 with Kre5 and Atg22 (A and B) The stability of Kre5-HA in YM27 (ROT1 KRE5-HA), YM28 (rot1-2 KRE5-HA), YM214 (ROT1 kar2-1), and YM215 (rot1-2 kar2-1) was examined by [35S]Met/Cys pulse-chase experiments as described for HA-Kre6 (Figure 4, A and B). A representative autoradiograph is shown in A. Kre5-HA radioactivity was quantified, and the averages and SDs of at least four independent experiments are shown in B. (C) Interaction between Rot1 and Kre5-HA. YM27 was incubated in YPD at 30°C and lysed, and IP using anti-Rot1 antibody or nonimmune serum (n.i.) was performed under nondenaturing conditions at 4°C. The immunoprecipitants were subjected to Western blotting to detect Kre5-HA and Rot1. (D) Cellular level of Atg22-HA is decreased by the rot1-2 mutation. ROT1 and rot1-2 cells expressing ATG22-HA from a centromeric plasmid (YM191 and YM192 [pRS314-ATG22-HA]) were incubated in SC at 23°C or shifted to 37°C for 6 h. The cells were lysed, and Atg22-HA and Sec61 were detected by Western blotting.