FIGURE 4.
Accumulating phosphorylated Ras1 in the proteasome-deficient mutants. A and B, a proteasome-deficient mutant (cim3-1 or rpn11S119A) and its isogeneic wild type were grown in YPD medium to mid-log phase. Whole cell extracts were resolved by 8% SDS-PAGE and probed with anti-Ras or anti-Pgk1 antibodies. Quantification of immunoblots by densitometry from three independent experiments is shown in the right panels. C, cells expressing either Ras1, Ras1S226A, or Ras1S226E under the control of the RAS1 promoter were grown to mid-log phase. Whole cell extracts were prepared, separated by sucrose gradient fractionation, resolved by 10% SDS-PAGE, and probed with anti-Ras, anti-Ste4, or anti-Pgk1 antibodies. The asterisks denote the position of Ras1 species with the same mobility on SDS-PAGE.