MIP1, A NEW YEAST GENE HOMOLOGOUS TO THE RAT MITOCHONDRIAL INTERMEDIATE PEPTIDASE GENE, IS REQUIRED FOR OXIDATIVE-METABOLISM IN SACCHAROMYCES-CEREVISIAE

Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochon drial intermediate peptidase (MIP) is typical of many mitochondrial pr ecursor proteins imported to the matrix and the inner membrane. We pre viously described the molecular characterization of rat liver MIP (RMI P) and indicated a putative homolog in the sequence predicted from gen e YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP) , which is 54% similar and 31% identical to RMIP and includes a putati ve 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH , while the predicted YCL57w protein contains a comparable sequence wi th a lower degree of homology. No obvious biochemical phenotype was ob served in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. Th e mip1 mutant exhibited defects of complexes III and TV of the respira tory chain, caused by failure to carry out the second MIP-catalyzed cl eavage of the nuclear-encoded precursors for cytochrome oxidase subuni t IV (CoxIV) and the iron-sulfur protein (Fe S) of the bc(1) complex t o mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted t o the inner membrane. Moreover, mip1 mitochondrial fractions failed to early out maturation of the human ornithine transcarbamylase intermed iate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence . Thus, YMIP is a functional homolog of RMIP and represents a new comp onent of the yeast mitochondrial import machinery.