Abstract
Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.
Highlights
Diates of the pathway are not detected in coq mutants (6 –12)
The coq10 mutant is similar to other coq mutants in which optimal oxidation of NADH and succinate by isolated mitochondria depends on addition of coenzyme Q2
BY⌬COQ10 and W303⌬COQ10 Phenotype—BY⌬COQ10 is a respiratory deficient mutant of the Genome Deletion Strain Collection. It was made by replacing reading frame YOL008W/COQ10 with the kanamycin-resistance cassette in strain BY4741. aW303⌬COQ10 and W303⌬COQ10 were obtained by replacing YOL008W with HIS3 in the wild type haploid W303-1A and W303-1B, respectively
Summary
Cloning of COQ2, COQ7, COQ8, and COQ10—Recombinant plasmids containing COQ2 (pG10/T1) and COQ7 (pG64/T2) and COQ8 (pG75/T2) were cloned by transformation of W303⌬COQ10 with a yeast genomic library constructed from nuclear DNA partially digested with a combination of BamHI and BglII and cloned in the yeast/Escherichia coli shuttle plasmid YEp352 [22]. The 1.3-kb fragment containing the COQ10 reading frame plus 700 bp of 5Ј and 50 bp of 3Ј-untranslated sequence was digested with HindIII and cloned in YEp352. Disruption of COQ10—The COQ10 gene and ϳ500 bp of upstream and downstream sequence was amplified with primers: 5Ј-ggcggatccttcatgattagctatagtacg and 5Ј-ggcggatccgtcctcccagtgttcatctgg. This fragment was digested with BamHI and cloned in pUC19 [24].
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