Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.
We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.
LaMarche
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AE
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755.
Abate
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MI
Chan
S H
SH
Trumpower
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BL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013057:Spectrum Analysis
D004271:DNA, Fungal; D005656:Fungal Proteins; D003576:Electron Transport Complex IV