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A conserved START domain coenzyme Q-binding polypeptide is required for efficient Q biosynthesis, respiratory electron transport, and antioxidant function in Saccharomyces cerevisiae - PubMed

. 2013 Apr;1831(4):776-791.

doi: 10.1016/j.bbalip.2012.12.007. Epub 2012 Dec 25.

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A conserved START domain coenzyme Q-binding polypeptide is required for efficient Q biosynthesis, respiratory electron transport, and antioxidant function in Saccharomyces cerevisiae

Christopher M Allan et al. Biochim Biophys Acta. 2013 Apr.

Abstract

Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain.

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Figures

Figure 1
Figure 1

Conserved amino acid residues in Coq10 polypeptide homologues. Protein sequences were aligned with BioEdit (

http://www.mbio.ncsu.edu/BioEdit/bioedit.html

) (Ibis Biosciences, Carlsbad, CA) and shaded as described by Genedoc with three levels of shading (

http://www.nrbsc.org/gfx/genedoc/

) [54]. Residues conserved in all proteins are shaded black, in 80% dark grey, and in 60% light grey. Amino-terminal segments of eukaryotic polypeptides preceding the first methionine of the C. crescentus (Ccr) and E. coli (Eco) polypeptides are not conserved and were omitted from the alignment for clarity. Residues determined to be important for maintenance of respiration are designated with filled symbols and include C. crescentus V70K (this work), S. pombe L63A and W104A [11]. Additional mutations affecting S. cerevisiae Coq10p function are designated by ‡ and include K50E, L96S, E105K, and K162D [18]. Mutation of K115E in CC1736 (marked by an open square) did not impair rescue of the S. cerevisiae coq10 null mutant (this work). The aligned sequences include: S. cerevisiae COQ10 (NCBI GeneID: 854154), Schizosaccharomyces pombe COQ10 (942096), C. crescentus CBL5 (942096), E. coli yfjG (945614), Homo sapiens COQ10A (93058), M. musculus COQ10B (67876), Drosophila melanogaster CG9410 (35568), Danio rerio Zgc:73324 (393762), Chlamydomonas reinhardtii COQ10 (5718019), and Arabidopsis thaliana AT4G17650 (827485).

Figure 2
Figure 2

Complementation of a yeast coq10 null mutant by expression of C. crescentus CC1736 requires the amino acid residue V70 for respiratory function. Wild-type, respiratory deficient cor1, yeast Q-less coq3 null mutant, and the coq10 null mutant were grown in SD complete medium and harvested during mid-log phase (0.2-1.0 OD600nm). The coq10 null mutant W303ΔCOQ10 was transformed with each of the following multi-copy plasmids: Empty (pRS426), COQ8 (p4HN4), COQ10 (pG140/ST3), CC1736 (pRCM-CC1736), or with plasmids encoding CC1736 with amino acid substitutions K115E (pRCM-K115E) or V70K (pRCM-V70K). Yeast transformants were grown in selective media and harvested during mid-log phase. Cells were washed twice with sterile water and diluted to a final OD600nm of 0.2. Serial dilutions (1:5) were prepared and 2 μl of each sample was spotted onto SD-Complete or SD-Ura, and rich glycerol (YPG) plate medium and incubated at 30 °C for 3 or 4 days, as indicated.

Figure 3
Figure 3

Mitochondria were isolated from wild type, respiratory deficient cor1 null mutant, Q-less coq3 null mutant, Q-replete coq10 null mutant and coq10 null mutant harboring plasmids expressing the designated proteins. NADH-cytochrome c reductase activity was determined in the absence (white bars) or presence (black bars) of 1 μM coenzyme Q2 (performed in triplicate for each sample). NADH-cytochrome c activity of the yeast coq3 null mutant, coq10 null mutant and coq10 null mutant expressing empty vector and V70K is significantly rescued by the addition of 1 μM coenzyme Q2; a, p < 0.0175; b, p < 6.1 E−04; c, p < 3.3 E−03. Values are given as the average ± standard deviation.

Figure 4
Figure 4

Yeast coq8, coq9, and coq10 mutants are hypersensitive to treatment with αLnn. Yeast strains were grown in YPD media and harvested during mid-log phase (0.2-1.0 OD600nm). The cor1 and atp2 null mutants serve as respiratory deficient controls. Cells were washed twice with sterile water and resuspended in phosphate buffer to a final OD600nm of 0.2. The designated fatty acids (final concentration of 200 μM) were added to a flask containing 20 ml of yeast /phosphate buffer as described in Section 2.6. Samples were removed either before addition of fatty acids (0 hour control) or after 2 hr of incubation at 30 °C in the presence of the designated fatty acid. Serial dilutions (1:5) were prepared and 2 μl were spotted onto YPD plate medium. Images were taken after two days of growth at 30 °C.

Figure 5
Figure 5

Yeast coq10 null mutants expressing Coq8p, Coq10p, or CC1736 are resistant to treatment with αLnn. The fatty acid sensitivity assay was performed as described in Fig. 4 except three 100 μL aliquots were removed at 9 h of either no treatment or 200 μM αLnn. Dilutions were prepared, and then spread onto SD-complete or SD−Ura plate medium. The chart shows the number of colony forming units (CFU) of untreated (black) and αLnn-treated (white) yeast cells. Yeast coq3Δ, coq10Δ, and coq10Δ null mutants harboring empty vector are profoundly sensitivity to PUFA treatment as compared to PUFA treated wild-type yeast as determined by the two-sample t test; a, p < 4.3 E−07. Yeast coq10 null mutants expressing Coq8p, Coq10p, or CC1736 are resistant to PUFA treatment as compared to coq10 null mutants expressing empty vector; b, p < 2.6 E−04.

Figure 6
Figure 6

Sensitivity of coq10 null mutant to αLnn treatment is due to increase levels of lipid peroxidation. (A) Wild-type cells were treated with αLnn for 2 hours and three 100 μl aliquots were removed and spread onto YPD plates after dilution. The chart shows the CFU per μl. (B) Following αLnn treatment, yeast cells were treated with 10 μM C11-Bodipy 581/591 for 30 min at room temperature. Four 100 μl aliquots were plated in a 96-well plate and the fluorescence was measured by fluorimetry. (C) Lipid peroxidation within cells was examined as described in (B) except cells were visualized by fluorescent microscopy. Green fluorescence indicates increased levels of lipid peroxidation. Scale bar = 6.6 μm.

Figure 7
Figure 7

Treatment of yeast coq10 null mutants with BHT, vitamin E, or vitamin C rescues the αLnn toxicity. The fatty acid sensitivity assay and statistical analyses were as described for Fig. 4, except yeast were treated with designated antioxidant compounds prior to the addition of αLnn. Three 100 μL aliquots were removed at 4 h, and following dilution, spread onto YPD plates. Cell viability was assessed by colony count. Yeast strains include wild type (white), cor1 (black), coq10 (light gray), or coq3 (dark gray). In the absence of antioxidants, the number of surviving αLnn treated coq3 and coq10 null mutant cells is significantly lower as compared to wild type and cor1 null mutants (a, p < 3.1 E−05). In the presence of lipid soluble antioxidants, the number of surviving αLnn treated coq3 and coq10 null mutant cells is significantly higher as compared to no antioxidant treatment (b, p < 2 E−05, c, p < 2.2 E−04). The water-soluble antioxidant, vitamin C failed to rescue coq3 mutant cells hypersensitivity to αLnn treatment (a, p < 3.1 E−05). In contrast, vitamin C afforded partial protection of the coq10 mutant cells hypersensitivity to αLnn treatment (d, p < 8 E−03).

Figure 8
Figure 8

Yeast coq10 null mutants have decreased de novo synthesis of 13C6-labeled Q6 compared to wild type, most notably during early-log phase growth. Wild-type and coq10 null yeast strains were cultured in SD-complete medium overnight and diluted to an OD600nm of 0.05 in DOD-complete medium. Ethanol (vehicle-control), 13C6-4HB (white bars), or 13C6-pABA (black bars) were added to yeast cultures at an OD600nm of 0.5 (early-log phase), 1.5 (late-log phase), or 3.0 (late-log phase). Prior to lipid extraction a known amount of Q4 was added as an internal standard to each sample and to the Q6 calibration standards. 13C6-labeled precursor-to-product ion transitions are as follows: (A) 13C6-HAB, 552.4/156.0; (B) 13C6-HHB, 553.4/157.0; (C) 13C6-IDMQ6, 566.6/172.0; (D) 13C6-DMQ6, 567.6/173.0; (E) 13C6-Q6, 597.4/203.1. Dashed arrows leading from HAB to IDMQ6 and from HHB to DMQ6 indicate multiple steps of Q biosynthesis (requiring Coq3, Coq4, Coq5, and Coq6). Solid arrows indicate the deimination of IDMQ6 to DMQ6 (requiring Coq9), and the final two steps converting DMQ6 to Q6 (requiring Coq7 and Coq3). Error bars depict the average ± standard deviation (n=4). (The total content of Q6 and Q6-intermediates (13C6-labeled + unlabeled), is depicted in Fig. S2). Statistical significance between pairs of samples was determined with the Student's t-test and lower-case letters above bars are indicative of statistical significance. In (A) and (B) the relative content of 13C6-labeled HAB or HHB in the coq10 null during early-, mid-, or late-log phase growth were compared to the corresponding growth phase of the wild type (a, p < 0.0001; b, p = 0.0001; c, p = 0.0112). The significance level α was adjusted to 0.0167 according to the Bonferroni correction for both (A) and (B). In (E) the content of 13C6-labeled Q6 in the coq10 null during early-, mid-, or late-log phase growth was compared to amounts present in the corresponding growth phase of the wild type (a, p ≤ 0.0004). Additionally 13C6-labeled Q6 in mid- and late-log phase was compared to early-log phase for both wild type and the coq10 null. Labeled 13C6-Q6 in wild type at late-log phase was significantly different compared to early-log phase (b, p ≤ 0.0004), and labeled Q6 in the coq10 null in mid-log phase was significantly different compared to early-log phase (c, p = 0.0008 for 13C6-pABA). For all analyses in (E) the significance level α was adjusted to 0.0033 according to the Bonferroni correction.

Figure 9
Figure 9

De novo synthesis of 13C6-Q6 in yeast coq10 null mutants is rescued upon transformation with COQ10, CC1736 or COQ8. The designated yeast strains were cultured in SD−Ura medium overnight, and diluted to an OD600nm of 0.05 in DOD−Ura medium. 13C6-4HB (white bars) or 13C6-pABA (black bars) was added to yeast cultures at an OD600nm of 0.5, corresponding to early-log phase. Lipid extraction of samples and analysis of precursor-to-product transitions was performed as described in the Fig. 8 legend. 13C6-Q6 and 13C6-labeled Q6-intermediates are shown as in Fig. 8. The bars depict the average ± standard deviation (n=4). (The total content of Q6 and Q6-intermediates (13C6-ring-labeled + unlabeled) is depicted in Fig. S3). Statistical significance between pairs of samples was determined with the Student's t-test and lower-case letters above bars are indicative of statistical significance. In (A) and (B) the relative content of 13C6-labeled HAB or HHB in each of the coq10 null transformants was compared to wild type (a, p < 0.0001). The relative content of 13C6-labeled HAB or HHB between the coq10 null with empty vector and the other coq10 null transformants was also compared (b, p ≤ 0.0003). In (A) and (B), the significance level α was adjusted to 0.005 according to the Bonferroni correction. In (E) the content of 13C6-labeled Q6 in each of the coq10 null transformants was compared to wild type (a, p < 0.0001). The relative content of 13C6-labeled Q6 between the coq10 null transformed with empty vector and the three other coq10 null transformants was also compared (b, p ≤ 0.0024). For all analyses in (E) the significance level α was adjusted to 0.005 according to the Bonferroni correction.

Figure 10
Figure 10

C. crescentus CC1736 binds Q10, Q2, Q3, and DMQ3. (A) Purified CC1736 (right panel) or cytochrome c (left panel) were added to binding buffer containing Q10 at the eight concentrations designated (0.01-0.18 mM). Samples were incubated for 45 min at 30 °C and unbound ligands were separated from the protein by application to a desalting column, and lipid extracts of the eluate were subjected to reversed-phase HPLC and the ligands were detected by UV absorbtion as described in section 2.13. (B) shows the amount of each ligand recovered in association with either CC1736 (black diamonds) or cytochrome c (open squares): ergosterol, Q10, Q2, Q3, DMQ3, or FHB in ascending concentration. One representative assay is shown of at least two assays performed for each ligand. Each concentration of ligand was tested in duplicate per binding assay.

Figure 11
Figure 11

A model for Coq10/START domain polypeptide function in de novo Q6 biosynthesis and in delivery of Q6 to respiratory chain complexes. (A) In wild-type cells, the Coq10:Q protein:ligand complex is postulated to deliver Q6 to the multisubunit Coq polypeptide complex and so enhance stability of the Coq polypeptides and de novo synthesis of Q6. Newly synthesized Q6 in the mitochondrial inner membrane is delivered to respiratory chain complexes and can function as an antioxidant. We postulate that Coq10:Q6 may also deliver Q6 to respiratory complexes. (B) The coq10 null mutant contains lower steady state levels of Coq polypeptides [20] and is shown with a less stable multisubunit Coq complex. Impaired de novo synthesis of Q6 and lack of Coq10:Q6 delivery to respiratory complexes accounts for the inefficient respiration observed in the coq10 null mutant. (C) The coq10 null mutant can be rescued by over-expression of COQ8, via its ability to restore the Coq polypeptide complex [25]. We postulate that the enhanced de novo Q6 biosynthesis formed by the Coq multisubunit complex is efficiently delivered to respiratory complexes, despite the absence of Coq10p.

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