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 of Saccharomyces 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 ironsulfur 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 and bc 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 the bc 1 complex.

The LYR Protein Mzm1 Functions in the Insertion of the Rieske Fe/S Protein in Yeast Mitochondria

“…Processing of the residual Rip1 by MPP and Oct1 appears normal in mzm1⌬ cells (Fig. 4A) (26). The Rip1 defect in mzm1⌬ cells is unlikely to be due to impaired Fe/S cluster formation based on previous studies.…”

“…The maturation of Rip1 into the bc 1 complex involves a series of steps. Rip1 is synthesized in the cytoplasm with an N-terminal mitochondrial targeting sequence that is proteolytically cleaved by the matrix processing protease (MPP) during im- The remaining steps in Rip1 maturation involve insertion of the 2Fe-2S cluster within the matrix, translocation of the Fe/S domain across the IM, and finally the formation of a disulfide bond (11,23,25,26). Processing of the residual Rip1 by MPP and Oct1 appears normal in mzm1⌬ cells (Fig.…”

Failure to Insert the Iron-Sulfur Cluster into the Rieske Iron-Sulfur Protein Impairs Both Center N and Center P of the Cytochrome bc 1 Complex

“…An S183A mutation that eliminated the hydrogen bond dropped the midpoint potential of the iron-sulfur protein from ϩ285 mV to ϩ150 mV and caused a 90% loss of activity. We also noted that when Ser-183 was replaced with Cys the resulting yeast were petite and the mitochondrial membranes had no bc 1 complex activity (6,9). However, we did not investigate the basis for loss of bc 1 complex activity.…”

“…We showed that the apoprotein can be assembled into the bc 1 complex if the cluster is not inserted (5), which suggests that the [2Fe-2S] cluster is added after the apoprotein is assembled into the bc 1 complex. We also showed that if processing of intermediate to mature size ironsulfur protein is blocked by mutations in the presequence, the intermediate size iron-sulfur protein accumulates in the bc 1 complex and is functionally active (6). This result establishes that the second protease-processing step takes place after intermediate size iron-sulfur protein has been assembled into the bc 1 complex and that the [2Fe-2S] cluster is inserted into the apoprotein before the second protease-processing step, although it does not establish that this sequence of events is obligatory.…”

“…The bc 1 complex from the CHS14 mutant lacks the 500 nm band (Fig. 2B), indicating that the cluster is absent and therefore explaining the absence of bc 1 complex activity in the mutant (6,9). Because the 500-nm CD band characteristic of the iron-sulfur cluster was not detectable, no attempt was made to characterize the cluster by EPR spectroscopy.…”