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Mitochondrial membrane biogenesis: Characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase
Author(s) -
McEwen Joan E.,
Cumsky Michael G.,
Ko Christine,
Power Scott D.,
Poyton Robert O.
Publication year - 1984
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240240305
Subject(s) - biology , protein subunit , mutant , microbiology and biotechnology , plasmid , gene , cytochrome c oxidase , scn3a , structural gene , mitochondrion , biochemistry , g alpha subunit
A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene ( COX5 ) by complementation. This mutant, E4‐238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild‐type polypeptide ( M r = 12,500), the polypeptides from E4‐238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis‐acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4‐238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13‐511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V ( COX5 ) and of the COX5 gene on a plasmid, together with the demonstration that plasmid‐encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.