The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector.
Phe82 is the most studied invariant residue of cytochrome c. However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e. using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector. Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level. Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon. The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast. All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c. However the F82C, F82P, and F82R variants grow at a significantly lower rate. After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified. It is concluded that all 20 amino acids are capable of supporting function. Reasons for the evolutionary invariance of Phe82 are discussed.
Hilgen
S E
SE
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Pielak
G J
GJ
D000483:Alleles; D001483:Base Sequence; D003062:Codon; D003090:Coliphages / Q000235:genetics; D003574:Cytochrome c Group / Q000235:genetics*; D045304:Cytochromes c; D005784:Gene Amplification; D005800:Genes, Fungal; D014644:Genetic Variation; D005822:Genetic Vectors; D005990:Glycerol / Q000494:pharmacology; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014170:Transformation, Genetic
C014059:CYC1 protein, S cerevisiae; D003062:Codon; D003574:Cytochrome c Group; D007773:Lactates; D029701:Saccharomyces cerevisiae Proteins; D019344:Lactic Acid; D010649:Phenylalanine; D045304:Cytochromes c; D005990:Glycerol