Processing of the Presequence of the Schizosaccharomyces pombe Rieske Iron-Sulfur Protein Occurs in a Single Step and Can Be Converted to Two-step Processing by Mutation of a Single Proline to Serine in the Presequence*

Abstract The iron-sulfur proteins of the cytochromebc 1 complexes of Schizosaccharomyces pombe and Saccharomyces cerevisiae contain the three amino acid motif RX(↓)(F/L/I)XX(T/S/G)XXXX(↓) that is typical for proteins that are cleaved sequentially in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). Despite the presence of this recognition sequence theS. pombe iron-sulfur protein is processed only once during import into mitochondria, whereas the S. cerevisiae protein is processed in two steps. Import of S. pombe iron-sulfur protein in which the putative MIP or MPP recognition sites are eliminated by site-directed mutagenesis and import of iron-sulfur protein into mitochondria from yeast mutants that lack MIP activity indicate that one step processing of the S. pombeiron-sulfur protein is independent of those sites and of MIP activity. Sequencing of the mature protein obtained after import in vitro and of the endogenous iron-sulfur protein isolated from mitochondrial membranes by preparative 2D-electrophoresis shows that MPP recognizes a second site in the presequence and processing occurs between residues 43 and 44. If proline-20 of the S. pombe presequence is changed into a serine, a second cleavage step is induced. Conversely, if serine-24 of the S. cerevisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-sulfur protein to accumulate. Together these results indicate that a single amino acid change in the presequence is responsible for one-step processing in S. pombe versustwo-step processing in S. cerevisiae.