FIGURE 3:
Cooperation of Ste23 and Cym1 in mitochondrial peptide degradation. (A) In vitro–synthesized Ste23 protein with a C-terminal hexahistidine tag (WT or E121Q mutation in the catalytic center) was incubated with Cox4 presequence peptide and amyloid β peptide (Aβ1–28) for indicated periods of time. Samples were analyzed by SDS–PAGE, followed by immunodecoration with Cox4 presequence specific antibodies and anti-histidine to detect Ste23. (B) Soluble extracts from WT and ste23Δ mitochondria were supplemented with Cox41–17 (lanes 1–8) and Sod21–18 (lanes 9–16) presequence peptides and incubated for indicated time. Peptide degradation was monitored by SDS–PAGE and immunodecoration. Mge1, loading control. (C) Double deletion of STE23 and CYM1 results in severe growth defect. Strains were grown under respiratory growth conditions (YPG) at 19°C. (D) Soluble mitochondrial fractions from cym1Δ and ste23Δcym1Δ yeast cells were incubated with Cox4 presequence peptides or amyloid β peptides for indicated time. Samples were analyzed by SDS–PAGE and immunodecoration. Mas1 served as loading control.