Intermediate length Rieske iron-sulfur protein is present and functionallyactive in the cytochrome bc(1) complex of Saccharomyces cerevisiae

To investigate the relationship between post-translational processing of th e Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly i nto the mitochondrial cytochrome bc(1) complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (M IP) had been destroyed. When yeast strain JPJ1, in which the gene for the i ron-sulfur protein is deleted, was transformed with these constructs on a s ingle copy expression vector, mitochondrial membranes and bc(1) complexes i solated from these strains accumulated intermediate length iron-sulfur prot eins in vivo. The cytochrome bc(1) complex activities of these membranes an d bc(1) complexes indicate that intermediate iron-sulfur protein (i-ISP) ha s full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP, When iron-sulfur protein is imported i nto mitochondria in vitro, i-ISP interacts with components of the bc(1) com plex before it is processed to m-ISP. These results establish that the iron -sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes p lace after i-ISP has been assembled into the bc(1) complex.