Import of the iron-sulfur protein of the cytochrome b.c1 complex into yeast mitochondria.
The yeast gene for the Rieske iron-sulfur protein of the cytochrome b.c1 complex was subcloned into the expression vector, pSP64, then transcribed and translated in vitro in a reticulocyte lysate in the presence of [35S]methionine. Import studies in vitro of the newly synthesized precursor form of the iron-sulfur protein into isolated yeast mitochondria revealed that the precursor form of the iron-sulfur protein is processed into the mature form via an intermediate form. After the import reaction at 18 or 27 degrees C, treatment of mitochondria with exogenous protease indicated that both intermediate and mature forms had been internalized into mitochondria where they were resistant to digestion by external protease. Import and processing of the iron-sulfur protein into mitochondria also occurred at temperatures ranging from 2 to 27 degrees C in a temperature-dependent manner. Processing of the precursor form to the intermediate form appeared to be less sensitive to temperature than the processing of the intermediate form to the mature form. Moreover, at temperatures of 12 degrees C or lower, the mature form produced was completely digested by exogenous protease suggesting that it was assembled incorrectly in the membrane and not assembled into the b.c1 complex. The successive disappearance of first the mature form and then the intermediate form of the iron-sulfur protein by increasing concentrations of the metal chelators, EDTA and o-phenanthroline, suggested that two different proteases requiring divalent metal ions are involved in the two-step processing of the presequence of the iron-sulfur protein. Furthermore, mitoplasts containing only the matrix/inner membrane fraction were able to import and process the precursor form of the iron-sulfur protein indicating that both proteolytic processing events occur in the matrix/inner membrane fraction.
Fu
W
W
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
Japa
S
S
Beattie
D S
DS
D014450:Electron Transport Complex III / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000235:genetics*; D008861:Microsomes / Q000201:enzymology*; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013816:Thermodynamics; D014158:Transcription, Genetic
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D011498:Protein Precursors; D014450:Electron Transport Complex III