PROCESSING OF THE PRESEQUENCE OF THE SCHIZOSACCHAROMYCES-POMBE RIESKEIRON-SULFUR PROTEIN OCCURS IN A SINGLE-STEP AND CAN BE CONVERTED TO 2-STEP PROCESSING BY MUTATION OF A SINGLE PROLINE TO SERINE IN THE PRESEQUENCE

The iron-sulfur proteins of the cytochrome be, complexes of Schizosacc haromyces pombe and Saccharomyces cerevisiae contain the three amino a cid motif RX(down arrow)(F/L/I)XX(T/S/G)XXXX(down arrow) that is typic al for proteins that are cleaved sequentially in two steps by matrix p rocessing peptidase (MPP) and mitochondrial intermediate peptidase (MI P). Despite the presence of this recognition sequence the S. pombe iro n-sulfur protein is processed only once during import into mitochondri a, whereas the S. cerevisiae protein is processed in two steps, Import of S. pombe iron-sulfur protein in which the putative MIP or MPP reco gnition sites are eliminated by site-directed mutagenesis and import o f iron-sulfur protein into mitochondria from yeast mutants that lack M IP activity indicate that one step processing of the S. pombe iron-sul fur protein is independent of those sites and of MIP activity. Sequenc ing of the mature protein obtained after import in vitro and of the en dogenous 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. ce revisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-s ulfur protein to accumulate. Together these results indicate that a si ngle amino acid change in the presequence is responsible for one-step processing in S. pombe versus two-step processing in S. cerevisiae.