Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. III. Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster.
We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines. These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein. We have also converted histidine 184 to arginine. This histidine is conserved only in respiring organisms. The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources. In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources. The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases. Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted. In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted. These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b. Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size. Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.
Graham
L A
LA
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Trumpower
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002872:Chromosome Deletion; D003580:Cytochromes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013367:Submitochondrial Particles / Q000201:enzymology
D003580:Cytochromes; D007506:Iron-Sulfur Proteins; D009838:Oligodeoxyribonucleotides; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III