title: Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins.
abstract: We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: Martina G
authors.initials: MG
authors.lastname: Ding
authors.forename: Christine A
authors.initials: CA
authors.lastname: Butler
authors.forename: Scott A
authors.initials: SA
authors.lastname: Saracco
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Fox
authors.forename: François
authors.initials: F
authors.lastname: Godard
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: di Rago
authors.forename: Bernard L
authors.initials: BL
authors.lastname: Trumpower
mesh_terms: D003470:Culture Media; D045303:Cytochromes b / Q000235:genetics* / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005285:Fermentation; D005800:Genes, Fungal; D005822:Genetic Vectors; D007438:Introns / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D000637:Transaminases / Q000378:metabolism
chemical_list: D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins; D045303:Cytochromes b; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV; D000637:Transaminases; C018731:acetylornithine transaminase; D014450:Electron Transport Complex III