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Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae - PubMed

️Thu Jan 01 2015

Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae

Christopher M Allan et al. J Biol Chem. 2015.

Abstract

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1-COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.

Keywords: Coenzyme Q; Immunoprecipitation; Mass Spectrometry (MS); Mitochondrial Metabolism; Protein Complex; Proteomics; Q Biosynthetic Intermediates; Saccharomyces cerevisiae; Ubiquinone; Yeast.

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Figures

**FIGURE 1.**
**Proposed coenzyme Q biosynthetic pathway in *S. cerevisiae*.** Yeast utilize either 4HB or pABA to synthesize the first lipid intermediates of the pathway, HHB or HAB, respectively, through the actions of Coq1 and Coq2. These intermediates then undergo a series of ring modifications catalyzed by the enzymes denoted above each step to yield Q. Two steps in the pathway, denoted with *question marks*, remain uncharacterized. The step at which the amino group is converted to the hydroxyl group remains unclear and is denoted by *dashed arrows*. Two late stage intermediates, DMQ₆ and IDMQ₆, are readily detected in yeast. The hexaprenyl moiety is abbreviated as R.

**FIGURE 2.**
**Expression of CNAP-tagged Coq3, Coq6, or Coq9 proteins preserves growth on a nonfermentable carbon source and *de novo* Q biosynthesis.** A, designated yeast strains were grown overnight in 5 ml of YPD, diluted to an A₆₀₀ of 0.2 with sterile PBS, and 2 μl of 5-fold serial dilutions were spotted onto each type of plate medium, corresponding to a final A₆₀₀ of 0.2, 0.04, 0.008, 0.0016, and 0.00032. Plates were incubated at 30 °C, and growth is depicted after 2 days for both YPD and SD−His and 3 days for YPG. B and C, total ¹³C₆-Q₆ (¹³C₆-Q₆ + ¹³C₆-Q₆H₂) (B) and total ¹²C-Q₆ (¹²C-Q₆ + ¹²C-Q₆H₂) (C) were measured in the designated yeast strains by HPLC-MS/MS. Yeast strains were grown overnight in 5 ml of SD-complete diluted to an A₆₀₀ of 0.05 in 50 ml of DOD-complete, labeled with either ¹³C₆-4HB or ¹³C₆-pABA at an A₆₀₀ of 0.5, and harvested after 3 h of labeling. Lipid measurements were normalized by the wet weight of extracted cells. Each *bar* represents the mean of four measurements from two biological samples with two injections each. *Error bars* represent standard deviations. Statistical significance was determined with the two-tailed Student's t test, and the *lowercase letters* above the *bars* are indicative of statistical significance. In B, the content of total ¹³C₆-Q₆ in CNAP3, CNAP6, CNAP9, and *coq8*Δ was compared with wild type with the corresponding ¹³C₆-labeled precursor; ¹³C₆-Q₆ synthesized from ¹³C₆-pABA in CNAP6 was 120% of wild type, whereas ¹³C₆-Q₆ synthesized from ¹³C₆-4HB in CNAP9 was 70% of wild type (a, p = 0.0052; b, p = 0.0018). In C, the content of total ¹²C-Q₆ in CNAP3, CNAP6, CNAP9, and *coq8*Δ was compared with wild type with the corresponding ¹³C₆-labeled precursor; accumulated ¹²C-Q₆ in CNAP6 was 80% of wild type under ¹³C₆-4HB labeling, 120% in CNAP6 under ¹³C₆-pABA labeling compared with wild type, and 80% of wild type in CNAP9 labeled with ¹³C₆-4HB (a, p = 0.0423; b, p = 0.0179; c, p = 0.0224). ND, not detected.

**FIGURE 3.**
**CNAP-tagged Coq proteins co-precipitate several other Coq proteins.** W303, CNAP3, CNAP6, and CNAP9 purified mitochondria (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification with Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting with antisera to the designated yeast polypeptides. 25 μg of mitochondria protein were analyzed for each strain (M), and 2.5% of the first anti-PC elution (E1) volume was loaded per strain (25 μl). *Arrows* denote each tagged protein in their respective blots. The predominant band in the Coq3 blot detected in the mitochondrial samples represents a background protein and not Coq3, accounting for its presence in CNAP3 mitochondria.

**FIGURE 4.**
**Tandem affinity-purified Coq complexes co-purify with Q₆ and late-stage Q₆ intermediates.** For each strain, aliquots of the anti-PC eluates (29% of E1 and 29% of E2) were combined and subjected to lipid extraction with methanol and petroleum ether. Lipid extracts were analyzed by HPLC-MS/MS for Q₆ (A), DMQ₆ (C), and IDMQ₆ (E). Measured lipids were normalized by the extracted eluate volume. *Bars* represent the means of two measurements, and *error bars* represent the standard deviation. Statistical significance was determined with the two-tailed Student's t test, and the *lowercase letters* above the *bars* are indicative of statistical significance. In A, the content of Q₆ in CNAP3, CNAP6, and CNAP9 was 11.7-, 8.6-, and 22.6-fold higher, respectively, compared with wild type (a, p = 0.0001; b, p = 0.0040; c, p = 0.0006). In C, the content of DMQ₆ in CNAP3, CNAP6, and CNAP9 was 30-, 24-, and 97-fold higher, respectively, compared with wild type (a, p = 0.0110; b, p = 0.0113; c, p = 0.0010). In E, there was no detectable IDMQ₆ in the wild type. ND, not detected. Representative overlaid traces of all four strains (W303, *purple*; CNAP3, *green*; CNAP6, *red*; CNAP9, *blue*) are shown for Q₆ (B), DMQ₆ (D), and IDMQ₆ (F).

**FIGURE 5.**
**SYPRO Ruby staining for total protein reveals unique bands in CNAP tagged eluates.** For each strain designated, aliquots of the anti-PC eluates (20% of E1 and 20% of anti-PC eluate two (E2)) were combined and dried with a SpeedVac at 60 °C for 2 h. Samples were resuspended in 42 μl of SDS sample buffer and separated with an 8–16% Criterion SDS-PAGE gel (Bio-Rad). Following separation proteins were fixed, and the gel was stained overnight with a 1:1 mixture of fresh and used SYPRO Ruby (Life Technologies). The gel was subsequently washed and visualized with an FX Pro Plus Molecular Imager (Bio-Rad) at 532-nm excitation, and emission was measured with a 555-nm long pass filter. The *ladder* denotes protein masses in kDa.

**FIGURE 6.**
**Yeast *ilv6*Δ and *ylr290c*Δ mutants retain the ability to grow on a nonfermentable carbon source.** Designated yeast strains were grown overnight in 5 ml of YPD and diluted to an A₆₀₀ of 0.2 with sterile PBS, and 2 μl of 5-fold serial dilutions were spotted onto each type of plate medium, corresponding to a final A₆₀₀ of 0.2, 0.04, 0.008, 0.0016, and 0.00032. Plates were incubated at 30 °C, and growth is depicted after 2 days for YPD and 3 days for YPG.

**FIGURE 7.**
**The yeast *ylr290c*Δ mutant, but not the *ilv6*Δ mutant, shows impaired *de novo* Q biosynthesis.** ¹³C₆-HAB (A), ¹²C-HAB (B), ¹³C₆-HHB (C), ¹²C-HHB (D), ¹³C₆-DMQ₆ (E), ¹²C-DMQ₆ (F), total ¹³C₆-Q₆ (¹³C₆-Q₆ + ¹³C₆-Q₆H₂) (G), and total ¹²C-Q₆ (¹²C-Q₆ + ¹²C-Q₆H₂) (H) were measured in the designated yeast strains by HPLC-MS/MS. Yeast strains were grown overnight in 5 ml of SD-complete, diluted to an A₆₀₀ of 0.05 in 50 ml of DOD-complete, labeled with either ¹³C₆-4HB or ¹³C₆-pABA at an A₆₀₀ of 0.5, and harvested after 3 h of labeling. Lipid measurements were normalized by the wet weight of extracted cells. Each *bar* represents the mean of four measurements from two biological samples with two injections each. *Error bars* represent standard deviations. Statistical significance was determined with the two-tailed Student's t test, and the *lowercase letters* above the *bars* are indicative of statistical significance. In A, the relative content of ¹³C₆-HAB in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p = 0.0017; b, p < 0.0001; c, p = 0.0014). In B, the relative content of ¹²C-HAB in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p < 0.0001; b, p = 0.0003; c, p = 0.0014; d, p = 0.0002). In C, the relative content of ¹³C₆-HHB in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p = 0.0001; b, p = 0.0003; c, p = 0.0005). In D, the relative content of ¹²C-HHB in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p = 0.0002; b, p = 0.0004; c, p = 0.0047; d, p < 0.0001). In E, the relative content of ¹³C₆-DMQ₆ in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p = 0.0441; b, p < 0.0001). ND, not detected. In F, the relative content of ¹²C-DMQ₆ in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor (a, p = 0.0453; b, p = 0.0002; c, p = 0.0003). ND, not detected. In G, the content of ¹³C₆-Q₆ in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor; the *ilv6*Δ mutant produced 60% ¹³C₆-Q₆ compared with wild type when labeled with ¹³C₆-4HB, and the *ylr290c*Δ mutant produced 6.1 and 4.5% ¹³C₆-Q₆ compared with wild type when labeled with ¹³C₆-4HB or ¹³C₆-pABA, respectively (a, p = 0.0017; b, p < 0.0001). ND, not detected. In H, the content of ¹²C-Q₆ in the three null mutants was compared with wild type with the corresponding ¹³C₆-labeled precursor; the *ylr290c*Δ mutant accumulated 17.5 and 21.8% ¹²C-Q₆ compared with wild type when labeled with ¹³C₆-4HB or ¹³C₆-pABA respectively (a, p < 0.0001). ND, not detected.

**FIGURE 8.**
**Expression of CNAP-tagged YLR290C preserves *de novo* Q biosynthesis.** Levels of total ¹³C₆-Q₆ (¹³C₆-Q₆ + ¹³C₆-Q₆H₂) (A) and total ¹²C-Q₆ (¹²C-Q₆ + ¹²C-Q₆H₂) (B) were measured in the designated yeast strains by HPLC-MS/MS. Yeast strains were grown overnight in 5 ml of SD-complete, diluted to an A₆₀₀ of 0.05 in 50 ml of DOD-complete, labeled with either ¹³C₆-4HB or ¹³C₆-pABA at an A₆₀₀ of 0.5, and harvested after 3 h of labeling. Lipid measurements were normalized by the wet weight of extracted cells. Each *bar* represents the mean of four measurements from two biological samples with two injections each. *Error bars* represent standard deviations. Statistical significance was determined with the two-tailed Student's t test, and the *lowercase letters* above the *bars* are indicative of statistical significance. In A, the content of ¹³C₆-Q₆ in CA-1 and *coq8*Δ was compared with wild type with the corresponding ¹³C₆-labeled precursor; ¹³C₆-Q₆ synthesized by CA-1 was 70% of wild type and 80% of wild type with ¹³C₆-4HB and ¹³C₆-pABA, respectively (a, p = 0.0005; b, p = 0.0196). ND, not detected. In B, the content of ¹²C-Q₆ in CA-1 and *coq8*Δ was compared with wild type with the corresponding ¹³C₆-labeled precursor; accumulated ¹²C-Q₆ in CA-1 was 70% of wild type when labeled with either ¹³C₆-4HB or ¹³C₆-pABA (a, p = 0.0202; b, p = 0.0001). ND, not detected.

**FIGURE 9.**
**Coq4, Coq5, and Coq7 co-precipitate with YLR290C-CNAP.** Purified mitochondria from W303 and CA-1 (15 mg of protein) were solubilized with digitonin and subjected to tandem affinity purification using Ni-NTA resin (Qiagen) followed by anti-PC-agarose (Roche). Samples were separated on 12% SDS-PAGE gels followed by transfer to PVDF membranes for immunoblotting. Mitochondria (25 μg of protein) (M) and 2.5% of the first anti-PC elution (E1) were analyzed for each of the two strains.

**FIGURE 10.**
**Tandem affinity-purified YLR290C-CNAP co-purifies with Q and late-stage Q intermediates.** For each strain, aliquots of the anti-PC eluates (29% of E1 and 29% of E2) were combined and subjected to lipid extraction with methanol and petroleum ether. Lipid extracts were analyzed by HPLC-MS/MS for total Q₆ (Q₆ + Q₆H₂) (A), DMQ₆ (C), and IDMQ₆ (E). Measured lipids were normalized by the extracted eluate volume. The *bars* represent the mean of two measurements, and the *error bars* represent the standard deviation. Statistical significance was determined with the two-tailed Student's t test, and the *lowercase letters* above the *bars* are indicative of statistical significance. In A, the content of Q₆ in CA-1 was 5.4-fold higher compared with wild type (a, p = 0.0418). In C, the content of DMQ₆ in CA-1 was 6.7-fold higher compared with wild type (a, p = 0.0025). In E, there was no detectable IDMQ₆ in the wild type. ND, not detected. Representative overlaid traces of both strains (W303, *magenta*; CA-1, *green*) are shown for Q₆ (B), DMQ₆ (D), and IDMQ₆ (F).

**FIGURE 11.**
**Sequence analysis of YLR290C and related proteins.** In A, YLR290C resides in a protein cluster populated by homologous fungal sequences including proteins that are fused to Coq10. Plant-like homologs form a distinct cluster composed of proteins from photosynthetic organisms. Prokaryotic sequences form a third independent cluster. The closest identified SDR subfamily distinct from YLR290C-like proteins includes human NDUFA9, a subunit of complex I, and orthologs in animals, fungi, and plants. In B, sequences representing each cluster from the protein similarity network in A were used to build a neighbor-joining tree. The tree is drawn to scale, with branch lengths in the same units (amino acid substitutions per site) as those of the evolutionary distances used to infer the phylogenetic tree. A branch length *scale bar* is shown below. A *number* after an organism name signifies the presence of multiple homologs in the tree. Protein identifications can be found in the
supplemental Data Set 1
. A schematic of the YLR290C-Coq10 fusion found in *U. maydis* is shown in C.

**FIGURE 12.**
**Amino acid alignment of selected fungal YLR290C fusion proteins with YLR290C and Coq10 from *S. cerevisiae*.** The N-terminal YLR290C-like portion is highlighted in *yellow*, and the Coq10 portion near the C terminus is highlighted in *blue*. The poorly conserved N-terminal sequence found in *P. antarctica* (res 1–100) is not shown. The *numbering* at the *top* of the alignment corresponds to the *P. antarctica* sequence. *Pan*, *P. antarctica*; *Sre*, *S. reilianum*; *Pfl*, *P. flocculosa*; *Mpe*, *M. pennsylvanicum*; *Uma*, *U. maydis*.

**FIGURE 13.**
**Model of the Q biosynthetic complex.** The organization of the complex is based on co-precipitation experiments performed in previous work (21) and this study, as well as two-dimensional blue native-PAGE analysis (28). Coq1, Coq2, and Coq10 have not been shown to associate with the complex. Coq10 binds Q and particular Q intermediates and is postulated to function as a Q chaperone for efficient respiration and *de novo* Q biosynthesis (56). Coq8 is required for the phosphorylation of Coq3, Coq5, and Coq7 (19), and co-precipitation of CNAP-tagged Coq6 demonstrates their physical association. Co-precipitation of tagged YLR290C, here designated Coq11, showed its association with Coq4, Coq5, and Coq7. The number of copies of each Coq polypeptide depicted in the CoQ-synthome is hypothetical because the stoichiometry has not been determined. Lipid analysis of co-precipitation eluates demonstrated the association of Q₆ and the late stage intermediates DMQ₆ and IDMQ₆ with the complex, illustrated as small molecules in association with Coq4.

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Identification of Coq11, a new coenzyme Q biosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae - PubMed