FIGURE 1:

Ygr031w/Imo32 is a novel Oct1 substrate with an unusual cleavage site motif. (A) Radiolabeled Imo32 precursor was incubated with mitochondria isolated from wild-type (WT) or oct1Δ yeast cells, treated with proteinase K, and analyzed by SDS–PAGE and digital autoradiography. Where indicated, the membrane potential (Δψ) was dissipated prior to the import reaction. prec. and p, precursor; i, intermediate; m, mature. (B) Import of [³⁵S]Imo32 into wild-type and mas1 mutant mitochondria. Samples were treated as in (A). (C) Radiolabeled Imo32 preprotein was incubated with isolated yeast mitochondria, and then treated with proteinase K. The samples, as well as ³⁵S-labeled truncated forms of Imo32, were analyzed by SDS–PAGE and digital autoradiography. (D) Import of [³⁵S]Imo32 with altered presequences into wild-type mitochondria. Cys-37 (corresponding to position −2 with regard to the proposed MPP cleavage site) was replaced by either alanine/A or aspartate/D. (E) Alignment of the cleavage motif of the 14 Oct1 substrate proteins from yeast. Characteristic three amino acid motif is highlighted in dark gray. Light gray denotes first amino acid of mature protein. Arrows indicate cleavage by MPP and Oct1, respectively. (F) Amino acid blot of relative frequency of the presequences (10 residues of the C-terminal segment) and first mature amino acids of the Oct1 substrate proteins identified in yeast (see Figure 1E). Arrows show cleavage sites of MPP and Oct1.