Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.
Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function.
Nakai
T
T
Meiji Institute of Health Science, Kanagawa, Japan.
Yasuhara
T
T
Fujiki
Y
Y
Ohashi
A
A
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D002455:Cell Division; D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004252:DNA Mutational Analysis; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D005990:Glycerol / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010101:Oxygen Consumption / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; D049069:ATP-Dependent Proteases; C082702:YME1 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D005990:Glycerol