FIGURE 2:

The metalloprotease Ste23 localizes to the mitochondrial matrix. (A) Radiolabeled Ste23 precursor was incubated with WT and tom22Δ mitochondria for various times. Where indicated, the membrane potential (Δψ) was dissipated before the import reaction. Samples were treated with PK and analyzed via SDS–PAGE and autoradiography. m, mature; p, precursor. (B) Import of [³⁵S]Ste23 precursor into mitochondria isolated from WT and mas1 temperature-sensitive mutant strains (Vögtle et al., 2009) after shifting of the culture for 6 h to 37°C. Impaired MPP activity in mas1 allows import but not presequence processing of Ste23 precursor (lane 4 vs. lane 3). Samples were treated as in A. (C) Mitochondria were subjected to hypoosmotic swelling (lanes 1–4), Triton X-100 treatment (lanes 5 and 6), or carbonate extraction at pH 11.5 (lanes 7 and 8). Samples were analyzed by SDS–PAGE and immunodecoration. Prot. K, Proteinase K; P, pellet; SN, supernatant.