Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector.
A phagemid (pING4) carrying the yeast iso-1-cytochrome c gene was constructed which bears all the elements necessary for replication in yeast and bacteria and may be converted into a single-stranded form of DNA for site-directed mutagenesis and nucleotide sequencing. The recombinant vector was used to create a complete set of 19 amino acid changes at position 82, a phylogenetically conserved phenylalanine residue in mitochondrial cytochrome c. All the different forms of cytochrome c were functional in vivo, based upon their ability to support respiration when the mutant proteins were expressed in a yeast strain (otherwise devoid of cytochrome c) grown on non-fermentable carbon sources, with only the strain containing the Cys82 variant having a substantially decreased growth rate. These results are interpreted in terms of the available structural and functional information previously reported on a subset of cytochrome c proteins with mutations at position 82.
Inglis
S C
SC
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Guillemette
J G
JG
Johnson
J A
JA
Smith
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D003090:Coliphages / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics*; D045304:Cytochromes c; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003062:Codon; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D010649:Phenylalanine; D045304:Cytochromes c