Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

Abstract

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.

Keywords: Coenzyme Q supplementation; Mitochondrial metabolism; Protein complex; Q-biosynthetic intermediate; Saccharomyces cerevisiae; Ubiquinone.

Figure 1

Q₆ biosynthesis in S. cerevisiae proceeds from either 4HB or pABA. The classic Q biosynthetic pathway is shown in blue emanating from 4HB (4-hydroxybenzoic acid). R represents the hexaprenyl tail present in Q₆ and all intermediates. The numbering of the aromatic carbon atoms used throughout this study is shown on the reduced form of Q₆, Q₆H₂. Coq1p synthesizes the hexaprenyldiphosphate tail, which is transferred by Coq2p to 4HB to form HHB (3-hexaprenyl-4-hydroxybenzoic acid). Alternatively, the red pathway indicates that pABA (para-aminobenzoic acid) is prenylated by Coq2p to form HAB (3-hexaprenyl-4-aminobenzoic acid). Both HHB and HAB are early Q₆-intermediates, readily detected in each of the coq null strains (Δcoq3-Δcoq9). Subsequent ring modification steps are thought to occur in the sequences shown, including hydroxylation by Coq6 in concert with ferredoxin (Yah1) and ferredoxin reductase (Arh1). Coq3 performs the two O-methylation steps, Coq5 the C-methylation step, and Coq7 performs the penultimate hydroxylation step. The functional roles of the Coq4, Coq8, and Coq9 polypeptides are elaborated in this study. Coq8p over-expression (hcCOQ8) in certain Δcoq strains leads to the accumulation of novel intermediates [17], suggesting these branched pathways. For example, in the presence of hcCOQ8, coq6 or coq9 mutants accumulate 4-AP (derived from pABA), and 4-HP (derived from 4HB) [17], indicating that in some cases decarboxylation and hydroxylation at position 1 of the ring may precede the Coq6 hydroxylation step. Purple dotted arrows designate the replacement of the C4-amine with a C4-hydroxyl and correspond to a proposed C4-deamination/deimination reaction, resulting in a convergence of the 4HB and pABA pathways. A putative mechanism to replace the C4-imino group with the C4-hydroxy group is shown in purple brackets for IDMQ₆ but could also occur on IDDMQ₆ (not shown). Several steps defective in the Δcoq9 strain are designated with red asterisks. Intermediates previously detected are shown in bold: 4-AP (3-hexaprenyl-4-aminophenol); DDMQ₆H₂, the reduced form of demethyl-demethoxy-Q₆; DMQ₆, demethoxy-Q₆; DMQ₆H₂, demethoxy-Q₆H₂ (the reduced form of DMQ₆); HHAB, 3-hexaprenyl-5-hydroxy-4-aminobenzoic acid; HMAB, 3-hexaprenyl-5-methoxy-4-aminobenzoic acid; 4-HP (3-hexaprenyl-4-hydroxyphenol); IDMQ₆, (4-imino-demethoxy-Q₆); IDMQ₆H₂, 4-amino-demethoxy-Q₆H₂ (the reduced form of IDMQ₆). Intermediates shown in parentheses indicate that they have not yet been detected: DHAP, 5-hydroxy-4-aminohexaprenylphenol; DHHB, 3-hexaprenyl-4,5-dihydroxybenzoic acid; DHHP, 4,5-dihydroxy-hexaprenylphenol; HMHB, 3-hexaprenyl-4-hydroxy-5-methoxybenzoic acid; IDDMQ₆H₂, 4-amino-demethyl-demethoxyQ₆H₂.

Figure 2

Coq2 is an integral membrane protein while Coq7 is peripherally associated to the inner mitochondrial membrane, facing toward the matrix side. (A), Mitochondria were subjected to a hypotonic swelling and centrifugation to separate intermembrane space protein (IMS) and mitoplasts. The mitoplasts were treated with 0.1 M Na₂CO₃, pH 11.5, or sonicated, then separated by centrifugation (100,000× g for 1 h) into supernatant (S) or pellet (P) fractions. (B), Intact mitochondria or mitoplasts were treated with 100 µg/ml Proteinase K for 30 min on ice, with or without detergent. Equal aliquots of pellet and TCA-precipitated soluble fractions were analyzed. Mitochondrial control markers are: Atp2, peripheral inner membrane protein; Cytb₂, inter-membrane space protein; Cytc₁, integral inner membrane protein; and Hsp60, soluble matrix protein.

Figure 3

Coq2 is resistant to salt extraction of sonicated mitoplasts while other Coq proteins are partially disassociated from the mitochondrial inner membrane. Purified mitochondria were subjected to hypotonic swelling to generate mitoplasts. Equal volumes of sonication buffer with or without salt were added to sonicated mitoplasts. Samples were incubated for 15 min on ice then separated by centrifugation (100,000 × g for 1 h) into supernatant or pellet fractions. Equal aliquots of pellet and TCA-precipitated supernatant fractions were analyzed.

Figure 4

Over-expression of Coq8 in the coq3 null mutant, but not in the coq4 null mutant, stabilizes the multi-subunit Coq polypeptide complex. Mitochondria were isolated from WT (W303-1A), coq3 null or coq4 null with and without the over-expression of Coq8 (hcCOQ8). Purified mitochondria (200 µg protein), were separated by two-dimensional blue native/SDS PAGE, and the immunoblots were probed with antibodies against Coq4 and Coq9. A sample of wild-type mitochondria separated just in the SDS-second dimension served as a positive control and is designated by M. Q or Q-intermediates derived from either 4HB or pABA that accumulated in the yeast strains were determined in the study by Xie et al. [17]. The coq mutants over-expressing Coq8 continue to produce HHB and HAB, but in addition the coq4 mutant also accumulates HHAB. Schematics show interactions of the Coq polypeptides and illustrate interactions potentially favored by over-expression of Coq8; ND, Coq polypeptides not detected.

Figure 5

Over-expression of Coq8 in coq5, coq6, coq7 or coq9 null mutant strains stabilizes the multi-subunit Coq polypeptide complex. Mitochondria were isolated from yeast strains harboring a deletion in one of the coq5, coq6, coq7, or coq9 genes with and without the over-expression of Coq8 (hcCOQ8). Purified mitochondria (200 µg protein) were separated by two-dimensional blue native/SDS PAGE, and the immunoblots were probed with antibodies against Coq4 and Coq9. A sample of wild-type mitochondria separated only in the SDS-second dimension served as a positive control and is designated by M. Q-intermediates derived from either 4HB or pABA that accumulated in the yeast mutants were determined in the study by Xie et al. [17]. The coq mutants over-expressing Coq8 continue to produce HHB and HAB, but in addition the designated late-stage Q-intermediates are also observed. Schematics show interactions of the Coq polypeptides and illustrate interactions potentially favored by over-expression of Coq8; dotted lines indicate that steady state-Coq polypeptides are present but are decreased relative to wild type; ND, Coq polypeptides not detected.

Figure 6

Over-expression of Coq8 in coq10 null mutant strain stabilizes the Coq4 and Coq9 polypeptide levels. Mitochondria were purified from coq10 null mutant yeast strain with and without the over-expression of Coq8 (hcCOQ8). (A), Purified mitochondria (20 µg protein) were subject to SDS-PAGE and Western blot probing with antibodies against Coq4, Coq9, and Porin. (B), Purified mitochondria (200 µg protein), were subjected to two-dimensional Blue Native/SDS PAGE, and immunoblots were probed with antibodies against Coq4 and Coq9. A sample of wild-type mitochondria separated only in the SDS-second dimension served as a positive control and is designated by M. The coq10 mutant produces Q₆ from 4HB and pABA and retains high molecular mass complexes of the Coq polypeptides as indicated by the schematic of the Coq complex; dotted lines indicate that steady state-Coq polypeptides are present but are decreased relative to wild type.

Figure 7

Inclusion of exogenous Q₆ during culture of coq1-coq9 null yeast mutants stabilizes certain Coq and mitochondrial polypeptides. Wild type or coq1-coq9 null mutant yeast were grown in 18 ml of YPD with either 1.5 µl ethanol/ml medium (no Q₆ addition) or the same volume of Q₆ dissolved in ethanol giving a final concentration of 10 µM Q₆ (+Q₆) for 42 hours. Yeast cells (10 A_600nm) were collected as pellets. Protein extracts were prepared from the pellets and analyzed by SDS-PAGE and immunoblot. Immunoblots were performed with antibodies against Coq1, Coq4, Coq7, Coq9, Atp2, malate dehydrogenase (Mdh1), or Rieske iron-sulfur protein (Rip1). Ponceau staining was used to detect the total proteins transferred to the membrane and served as the loading control.

Figure 8

Exogenous Q₆ increases synthesis of demethoxy-Q₆ (DMQ₆) in coq7 null mutant. Yeast coq7 null mutant was cultured in YPD with either 10 µg/ml ¹³C₆ -4HB and 1.5 µl ethanol/ml medium (no Q₆ addition) or 10 µg/ml ¹³C₆ -4HB and the same volume of Q₆ dissolved in ethanol giving a final concentration of 10 µM Q₆ (+Q₆) for 42 hours. Yeast cells (30 A_600nm) were collected as pellets and washed twice with distilled water. Q₄ (145.4 pmol) was added as internal standard. Lipid extracts were prepared from the pellets and analyzed by RP-HPLC-MS/MS. Multiple reaction monitoring (MRM) detected precursor-to-product ion transitions 567.0/173.0 (¹³C₆ -DMQ₆). The green trace designates the ¹³C₆-DMQ₆ signal in the +Q₆ condition, and the blue trace indicates the ¹³C₆-DMQ₆ signal in the absence of added Q₆. The peak areas of ¹³C₆-DMQ₆ normalized by peak areas of Q₄ are 0.0665 in coq7Δ +¹³C₆-4HB and 0.215 in coq7Δ+ Q₆ +¹³C₆-4HB.

Figure 9

Exogenous Q₆ increases the accumulation of 3-hexaprenyl-4-amino-5-hydroxybenzoic acid (HHAB) in coq4 and coq6 null mutants. Yeast coq4 and coq6 null mutants were cultured in YPD with either 1.5 µl ethanol/ml medium (no Q₆ addition) or the same volume of Q₆ dissolved in ethanol giving a final concentration of 10 µM Q₆ (+Q₆) for 42 hours. Yeast cells (30 A_600nm) were collected as pellets and washed twice with distilled water. Q₄ (145.4 pmol) was added as internal standard. Lipid extracts were prepared from the pellets and analyzed by RP-HPLC-MS/MS. Multiple reaction monitoring (MRM) detected precursor-to-product ion transitions 562.0/166.0 (HHAB) and 455.4/197.0 (Q₄). The arbitrary units (cps) and the scale is the same for all the traces within the same panel. In panels A and B, green traces designate the HHAB signal in the +Q₆ condition, and purple traces the HHAB signal in the absence of added Q₆. The peak areas of HHAB normalized by peak areas of Q₄ are 0.01 in coq4Δ (A), 0.14 in coq4Δ+ Q₆ (A), 0.003 in coq6Δ (B), and 0.03 in coq6Δ+Q₆ (B). The retention times of HHAB are 2.69 min in coq4Δ+Q₆ (A), and 2.71 min in coq6Δ+Q₆ (B).

Figure 10

Exogenous Q₆ increases the accumulation of 3-hexaprenyl-4-aminophenol (4-AP) in the coq6 null mutant. Lipid extracts were prepared from the cell pellets of coq6 null mutant yeast following growth in YPD with either the presence (+Q₆) or absence of Q₆ and analyzed by RP-HPLC-MS/MS as described in Fig.9. MRM detected precursor-to-product ion transitions 518.4/122.0 (4-AP) and 455.4/197.0 (Q₄). In panel A, the green trace designates the 4-AP signal in the +Q₆ condition, and the purple trace designates the 4-AP signal in the absence of added Q₆ (coq6Δ). The peak areas of 4AP normalized by peak areas of Q₄ are 0.008 in coq6Δ and 2.68 in coq6Δ+Q₆. Panel B shows the fragmentation spectrum for the 4-AP [M+H]⁺ precursor ion (C₃₆H₅₆NO⁺; monoisotopic mass 518.4), the 4-AP tropylium ion [M]⁺ (C₇H₈NO⁺; 122.06), and the 4-AP chromenylium ion [M]⁺ (C₁₀H₁₂NO⁺; 162.1).

Figure 11

Exogenous Q₆ leads to the accumulation of imino-demethoxy-Q₆ (IDMQ₆) in the coq9 null mutant. Lipid extracts were prepared from cell pellets of the coq9 null mutant yeast following growth in YPD with either the presence (+Q₆) or absence of Q₆ and analyzed by RP-HPLC-MS/MS as described in Fig. 8. Panel A shows the MRM detected precursor-to-product ion transition 560.5/166.2 (IDMQ₆). Panel B, shows the fragmentation spectrum for the IDMQ₆ [M+H]⁺ precursor ion (C₃₈H₅₈NO⁺; monoisotopic mass 560.4), the IDMQ₆ tropylium ion [M]⁺ (C₉H₁₂NO₂ ⁺; 166.1), and the IDMQ₆ chromenylium ion [M]⁺ (C₁₂H₁₆NO₂ ⁺; 206.1).

Figure 12

Proposed model for the yeast CoQ-synthome. This model is consistent with co-precipitation studies in S. cerevisiae with tagged Coq polypeptides, and the association of several of the Coq polypeptides in high molecular mass complexes, as assayed in digitonin extracts of mitochondria separated by two-dimensional blue native/SDS PAGE. The over-expression of Coq8, a putative kinase, is required to observe phosphorylated forms of Coq3, Coq5, and Coq7 [31]. Coq10, a START domain polypeptide, binds to Q and is postulated to act as a Q chaperone that delivers Q to the CoQ-synthome and/or the bc₁ complex [38]. Coq4 is denoted as a scaffolding protein, with binding sites for Q or polyisoprenyl-intermediates and serves to organize the high molecular mass Q biosynthetic complexes. See text for additional explanation.