Pseudo-wild type revertants from inactive apocytochrome b mutants as a tool for the analysis of the structure/function relationships of the mitochondrial ubiquinol-cytochrome c reductase of Saccharomyces cerevisiae.
We have analyzed the structure/function relationships of the yeast mitochondrial cytochrome b with a new methodology based upon the isolation of pseudo-wild type revertants from well-characterized cytochrome b respiratory deficient mutants. Our goal was to determine how cytochrome b function could be restored in such mutants, at least to some degree, by suppressor mutations within the protein. True wild type revertants were differentiated from pseudo-wild type revertants by the use of a simple and rapid screening technique based upon oligonucleotide hybridization. This can easily be used to analyze a large number of revertants. The suppressor mutations responsible for the restoration of respiratory competence were identified by sequencing the revertant's cytochrome b mRNA in crude mitochondrial RNA preparations. Using this new method we have analyzed 210 independent revertants. We report here nine novel cytochrome b structures conferring a variety of respiratory sufficient phenotypes, obtained from five respiratory deficient mutations affecting a short region of the protein (positions 131-138 of the polypeptide chain), presumably belonging to the ubiquinol oxidizing center of the bc1 complex.
di Rago
J P
JP
Centre de Génétique Moléculaire, Université P. et M. Curie, Gif-sur-Yvette, France.
Netter
P
P
Slonimski
P P
PP
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism; D045303:Cytochromes b; D014450:Electron Transport Complex III / Q000378:metabolism*; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D012331:RNA, Fungal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013367:Submitochondrial Particles / Q000378:metabolism
D001059:Apoproteins; D003573:Cytochrome b Group; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D045303:Cytochromes b; D014450:Electron Transport Complex III