Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

Abstract

COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q2. Rescue of respiration by Q2 is a characteristic of mutants blocked in coenzyme Q6 synthesis. Unlike Q6 deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q6. The physiological significance of earlier observations that purified Coq10p contains bound Q6 was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q2. This suggests that in vivo binding of Q6 by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.

Overexpression of the Coq8 Kinase in Saccharomyces cerevisiae coq Null Mutants Allows for Accumulation of Diagnostic Intermediates of the Coenzyme Q6 Biosynthetic Pathway

“…Indeed, most Cox proteins are degraded in the absence of any of the three mitochondrially encoded core subunits Cox1-Cox3 (27). Our present study establishes that Coq8 OE stabilizes the levels of Coq4, Coq6, Coq7, and Coq9 in ⌬coq3-⌬coq7 strains and therefore generalizes to most Coq polypeptides this stabilizing effect that had been described previously for Coq4 (17). The stabilized proteins are active because they allowed accumulation of novel Q 6 biosynthetic intermediates in several ⌬coq strains, suggesting that the Q 6 biosynthetic complex is at least partially assembled.…”

“…The effect of Coq8 OE is likely post-transcriptional because COQ4 mRNA levels in the ⌬coq7 strain were not dependent on the level of Coq8 OE (16). Recently, the low steady state level of Coq4 encountered in ⌬coq2, ⌬coq3, ⌬coq5, and ⌬coq7 strains was shown to be restored to wild-type levels by Coq8 OE (17). In the case of a ⌬coq6 strain, Coq8 OE allowed the specific accumulation of 3-hexaprenyl-4-hydroxyphenol (4-HP) ( Fig.…”

Aep3p-dependent translation of yeast mitochondrialATP8

“…Approximately 6000 respiratory-deficient mutants were pooled and transformed with pATP8-22, a centromeric TRP1 -based plasmid containing nATP8 (recoded for allotopic expression) under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter (GDP; Zampol et al , 2010). This plasmid is expected to complement mutants that cannot express mitochondrial ATP8 .…”

Coq6 Hydroxylase: Unmasked and Bypassed

“…The authors use a combination of yeast genetics, lipid biochemistry and clever feeding experiments of alternate ring precursors to characterize the defective step in yeast coq6 mutants. The authors capitalize on the observations that several of the yeast coq null mutants harboring multi-copy COQ8 have restored steady state levels of the Coq3 and Coq4 polypeptides (Zampol, et al, 2010), and in the case of the coq7 null, accumulate DMQ 6 , an intermediate just two steps removed from Q (Padilla, et al, 2009). Ozeir et al (2011) show: (1) the yeast coq6 null mutants harboring the COQ8 gene on a multi-copy plasmid produce intermediates lacking the C5-ring hydroxyl group; and (2) yeast coq6 null mutants expressing inactive Coq6p with two amino acid substitution mutations (Coq6-G386A,N388D) also accumulate the same two intermediates lacking the C5-ring hydroxyl, namely, 3-hexaprenyl-4-aminophenol (4-AP) and 3-hexaprenyl-4-hydroxyphenol (4-HP) (Fig.…”