Fig. 2. The HTM1 gene does not influence the degradation of mutant Sec61-2 protein. (A) Δmns1 and Δhtm1 do not suppress temperature-sensitive growth of sec61-2 mutant cells. Isogenic sec61-2 single- and double-mutant cells were plated in serial dilutions on rich medium and grown at 30 and 37°C for 3 days. (B) Degradation of Sec61-2 protein is not affected in mns1- and htm1-deficient cells. Cells were grown at 30°C to mid-log phase and shifted to 37°C for 2 h. Equal cell numbers were incubated with 100 µg/ml cycloheximide, aliquots were removed and cells broken at given time points. Extracted proteins were separated by SDS–PAGE, and analyzed and quantified by immunoblotting using anti-Sec61 antiserum. The protein quantity of time point 0 (steady-state level) was set to 100%.