Intermediate Length Rieske Iron-Sulfur Protein Is Present and Functionally Active in the Cytochrome bc 1Complex of Saccharomyces cerevisiae

Abstract To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein ofSaccharomyces cerevisiae and its assembly into 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 (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes andbc 1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc 1 complex activities of these membranes and bc 1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has 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 into mitochondria in vitro, i-ISP interacts with components of the bc 1 complex 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 place after i-ISP has been assembled into thebc 1 complex.