Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway

Abstract

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

FIGURE 1.

S. cerevisiae Q₆ biosynthetic pathway. Accumulation of Q₆ biosynthetic intermediates is caused by the overexpression of Coq8 in Δcoq strains. The classic Q biosynthetic pathway is shown in path 1 emanating from 4-HB. Coq1 (not shown) synthesizes the hexaprenyl-diphosphate tail that is transferred by Coq2 to 4-HB to form HHB. R represents the hexaprenyl tail present in all intermediates from HHB to Q₆. Alternatively, in path 2, pABA is prenylated by Coq2 to form HAB. Both HHB and HAB are early Q₆ intermediates, readily detected in each of the coq null strains (Δcoq3-Δcoq9). The numbering of the aromatic carbon atoms used throughout this study is shown on the reduced form of Q₆, Q₆H₂. Coq8 OE in certain Δcoq strains leads to the accumulation of the following compounds: 4-AP, 3-hexaprenyl-4-aminophenol; 4-HP, 3-hexaprenyl-4-hydroxyphenol; HHAB, 3-hexaprenyl-4-amino-5-hydroxybenzoic acid; HMAB, 3-hexaprenyl-4-amino-5-methoxybenzoic acid; DDMQ₆H₂, is the reduced form of demethyl-demethoxy-Q₆; IDMQ₆, 4-imino-demethoxy-Q₆; IDMQ₆H₂, 4-amino-demethoxy-Q₆H₂; DMQ₆, demethoxy-Q₆; DMQ₆H₂, demethoxy-Q₆H₂. IDDMQ₆H₂, 2-demethyl-4-amino-demethoxy-Q₆H₂, and DHHB, 3-hexaprenyl-4,5-dihydroxybenzoic acid, are shown in parentheses to indicate that they have not been detected in this study. Black dotted arrows (from path 2 to path 1) designate the replacement of the C4-amine with a C4-hydroxyl and correspond to the C4-deamination/deimination reaction. A putative mechanism to replace the C4-imino group with the C4-hydroxy group is shown in blue brackets for IDMQ₆ but could also occur on 2-demethyl-4-amino-demethoxy-Q₆ (IDDMQ₆) (not shown). 4-AP and 4-HP, which are formed upon inhibition of the C5-hydroxylation catalyzed by Coq6, are shown in red. Analogues of 4-HB and pABA allowing the bypass of certain steps in Q biosynthesis are indicated in green. Steps defective in the Δcoq9 strain are designated with a red asterisk.

FIGURE 2.

Overexpression of Coq8 in several Δcoq strains restores steady state levels of Coq4, Coq7, and Coq9. Whole cell lysates were prepared from W303-1B wild-type yeast (WT) or from the indicated Δcoq strains (all except Δcoq9 in W303 genetic background) with (+) or without (−) p4HN4, a multicopy plasmid with yeast COQ8 (Coq8). Yeast cells cultured in YPGal medium to mid-log (2–3 A_{600 nm}) were harvested, and aliquots of 20 A_{600 nm} were lysed and analyzed by SDS-PAGE (10% acrylamide) followed by transfer to PVDF membrane and immunoblotting with antibodies to the designated Coq polypeptides or to Pda1, the α-subunit of pyruvate dehydrogenase. A, Pda1 serves as a loading control; B, Coq1 and/or Coq5 serve as loading controls.

FIGURE 3.

Overexpression of Coq8 restores steady state levels of Coq polypeptides in isolated mitochondria. Isolated mitochondria were purified with Opti-Prep gradients from W303-1B wild-type yeast (WT) or from the designated Δcoq strains (W303 genetic background) with (+) or without (−) p4HN4 (Coq8). Aliquots of mitochondrial protein (20 μg) were analyzed by SDS-PAGE followed by immunoblot analyses with antisera to the designated Coq polypeptides (A) or with antisera to Coq6 (B). Coq1 serves as a loading control.

FIGURE 4.

Overexpression of Coq8-G130D does not restore steady state levels of Coq polypeptides. Whole cell lysates were prepared as described in Fig. 2 from W303-1B wild-type yeast (WT) or from Δcoq6 or Δcoq7 strains (W303 genetic background) harboring either no plasmid; pFL44, a multicopy plasmid expressing Coq8 (hcCoq8); G130D, a multicopy plasmid with Coq8 containing the G130D mutation (G130D); or p3HN4, a low copy plasmid expressing Coq8 (lcCoq8). Yeast cells were cultured in SD−Ura medium to mid-log (2–3 A_{600 nm}) phase and harvested, and aliquots of 20 A_{600 nm} were lysed and subjected to immunoblotting analyses with antisera to the designated Coq polypeptides. Pda1 serves as a loading control.

FIGURE 5.

Δcoq7 and Δcoq5 strains overexpressing Coq8 accumulate the respective Q intermediates, DMQ₆ and DDMQ₆. A, Δcoq7 cells (W303 genetic background) transformed with pFL44, an episomal vector encoding Coq8 (Coq8), were grown overnight in YNB-pABA-folate 2% dextrose with no additions or with 5 μm 4-HB or pABA. Lipid extracts of 4 mg of cells (no addition) or of 1.5 mg of cells (4-HB, pABA) were analyzed by HPLC-ECD. The peaks corresponding to DMQ₆ and to the Q₄ standard are marked. B, Δcoq5 cells (W303 genetic background) transformed either with pFL44 (Coq8) or an empty vector (vec) were grown overnight in YNB−pABA−folate 2% dextrose containing either no addition or 5 μm 4-HB or pABA. Lipid extracts of 10 mg of cells were analyzed by HPLC-ECD. C, Δcoq5 cells (W303 genetic background) containing either no plasmid or p4HN4, an episomal vector encoding Coq8 (Coq8), were first grown in YPGal + 0.1% dextrose and then cultured in DOGAL + 0.1% dextrose−pABA−folate−tyrosine overnight. Finally cells were collected and transferred to fresh DOGAL + 0.1% dextrose−pABA−folate in the presence of either ¹³C₆-4HB or ¹³C₆-pABA (10 μg/ml; 3 ml) and incubated for 2 h. Cells were collected (150 A_{600 nm}), and lipid extracts were subjected to RP-HPLC-ESI/MS-MS as described under “Experimental Procedures,” and detection of the precursor-to-product ion transition (553.4/159.0) was performed with MRM. The traces indicate arbitrary units, and the scale is the same for all traces within a panel.

FIGURE 6.

Bypass of the respiratory growth defect of the Δcoq7 strain with alternate ring precursors. A, Δcoq7 cells (W303 genetic background) transformed with an episomal vector coding for Coq8 (pFL44) were grown in YNB−pABA−folate 2% dextrose containing 1 mm 2,4-dihydroxybenzoic acid (2,4-diHB) or not (−). Lipid extracts of 2 mg of cells were analyzed by HPLC-ECD. B, WT W303 cells or Δcoq7 cells transformed either with an empty vector (vec), an episomal vector (pFL44) encoding Coq8 or Coq8-G130D, or with an episomal vector encoding Coq7 were grown in YNB−pABA−folate 2% dextrose for 24 h, and serial dilutions were spotted onto agar plates. The plates contained either YP 2% dextrose (Glu) or synthetic medium−pABA−folate supplemented with 2% lactate-2% glycerol (LG) containing either 4-HB or 2,4-dihydroxybenzoic acid (2,4-diHB) at 1 mm. The plates were incubated for 2 days (Glu) or 4 days (LG) at 30 °C.

FIGURE 7.

Δcoq3 and Δcoq4 strains overexpressing Coq8 or a coq4-1 strain accumulate early Q₆ biosynthetic intermediates. A, yeast Δcoq3 (BY4741 genetic background) and Δcoq4 cells (BY4742 genetic background) transformed with pFL44, an episomal vector encoding Coq8 (Coq8), were grown in YNB−pABA−folate 2% dextrose containing 100 μm VA or 100 μm 4-HB. Lipid extracts of 4 mg of cells were analyzed by HPLC-ECD. B, yeast Δcoq4 cells (W303 genetic background) transformed with p4HN4, an episomal vector encoding Coq8, and coq4-1 cells were grown in YPGal + 0.1% dextrose overnight and labeled in DOGAL + 0.1% dextrose−folate−pABA in the presence of either ¹³C₆-4HB or ¹³C₆-pABA (20 μg/ml; 3 ml) for 2 h. Lipid extracts of 100 A_{600 nm} of cells were analyzed by RP-HPLC-MS/MS as described under “Experimental Procedures,” and detection of the precursor-to-product ion transition (568.0/172.0) was performed with MRM. C, yeast coq4-1 mutant cells or wild-type yeast cells were grown in YPGal + 0.1% dextrose overnight and transferred into 3 ml of fresh DOGAL + 0.1% dextrose−folate−pABA in the presence of 100 μg/ml 4-amino-3-methoxybenzoic acid (AMB). Lipid extracts of 100 A_{600 nm} of cells were analyzed by RP-HPLC-ESI/MS-MS as described under “Experimental Procedures,” and detection of the precursor-to-product ion transition (576.0/180.0) was performed with MRM.

FIGURE 8.

Coq9 is important for Coq6 and Coq7 hydroxylation steps and for removal of the amino substituent. A, Δcoq9 cells (BY4742 genetic background) transformed either with an episomal vector coding for Coq8 (pFL44) or an empty vector (vec) were grown in YNB−pABA−folate 2% dextrose containing or not 5 μm 4-HB or pABA. Lipid extracts of 10 mg of cells were analyzed by HPLC-ECD. The peaks corresponding to DMQ₆, to IDMQ₆, or to the oxidized forms of 4-AP and of 4-HP are marked. B–E, yeast Δcoq6, Δcoq7, Δcoq9, in the absence or presence of p4HN4 (an episomal plasmid encoding Coq8) were cultured and prepared as described in Fig. 5. Yeast cell pellets (150 A_{600 nm} of cells) were subjected to RP-HPLC-MS/MS as described under “Experimental Procedures,” and detection of the designated precursor-to-product ion transitions (525.4/129.0, 524.4/128.0, 567.6/173.0, 566.6/172.0) was performed with MRM.