title: Processing of the intermediate form of the iron-sulfur protein of the BC1 complex to the mature form after import into yeast mitochondria.
abstract: The Rieske iron-sulfur protein of the cytochrome bc1 complex is synthesized in the cytosol as a precursor with an additional 30 amino acids at the amino terminus. After import into the mitochondrial matrix, the precursor is processed to the mature form by two distinct proteolytic cleavages. Addition of 2.5 mM EDTA and 0.5 mM o-phenanthroline to the incubation mixture during import of the iron-sulfur protein precursor in vitro resulted in the selective inhibition of the second processing step with the concomitant accumulation of the intermediate form. The intermediate form was chased to the mature form in the presence of antimycin and oligomycin (to block the formation of a membrane potential) provided that 0.5 nM ATP and a metal ion such as Ca2+, Mn2+, or Mg2+ were added. Ca2+ ion was the most effective and at a concentration of 2.5 mM resulted in the complete cleavage of the intermediate to the mature form. Addition of Zn2+, Co2+, Mo2+, and Fe2+ was not effective in restoring the second cleavage. The pH optimum for the processing of the intermediate form of the iron-sulfur protein to the mature form was between 6.8-8.0. Processing of the intermediate form of the iron-sulfur protein to the mature form was observed at temperatures ranging from 12 to 27 degrees C in a temperature-dependent manner. The time course during the chase indicated that the second processing step was completed within 2 min after addition of Ca2+ ions. Attempts to isolate the second processing enzyme by sonication of mitochondria or by solubilization with detergents such as digitonin, Triton X-100, dodecyl-maltoside, or octyl-glucoside were unsuccessful as only the first cleavage was observed. Hence, the second processing enzyme may be present in the inner membrane or matrix in a conformation disrupted by detergents or alternatively the enzyme may be very labile.
authors.affiliation: Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
authors.forename: R S
authors.initials: RS
authors.lastname: Ramabadran
authors.forename: D S
authors.initials: DS
authors.lastname: Beattie
mesh_terms: D003600:Cytosol / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D014450:Electron Transport Complex III / Q000096:biosynthesis / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D010618:Phenanthrolines / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D007506:Iron-Sulfur Proteins; D008670:Metals; D010618:Phenanthrolines; C037989:Rieske iron-sulfur protein; D004492:Edetic Acid; D014450:Electron Transport Complex III; D008274:Magnesium; C025205:1,10-phenanthroline