Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector |
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Authors: | Inglis Stephen C; Guillemette JGuy; Johnson Jeanette A; Smith Michael |
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Affiliation: | Department of Biochemistry and the Biotechnology Laboratory, University of British Columbia Vancouver, British Columbia, Canada V6T 1W5
1Present address Immunology Ltd 184 Cambridge Science Park, Milton Road, Cambridge CB4 4GN, UK |
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Abstract: | A phagemid (pING4) carrying the yeast iso-1-cytochrome c genewas constructed which bears all the elements necessary for replicationin yeast and bacteria and may be converted into a single-strandedform of DNA for site-directed mutagenesis and nucleotide sequencing.The recombinant vector was used to create a complete set of19 amino acid changes at position 82, a phylogenetically conservedphenylalanine residue in mitochondrial cytochrome c. All thedifferent forms of cytochrome c were functional in vivo, basedupon their ability to support respiration when the mutant proteinswere expressed in a yeast strain (otherwise devoid of cytochromec) grown on non-fermentable carbon sources, with only the straincontaining the Cys82 variant having a substantially decreasedgrowth rate. These results are interpreted in terms of the availablestructural and functional information previously reported ona subset of cytochrome c proteins with mutations at position82. |
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Keywords: | expression/ iso-1-cytochrome c/ mutagenesis/ protein function |
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