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Originally published In Press as doi:10.1074/jbc.M313712200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 11, 10052-10059, March 12, 2004
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Yeast Coq5 C-Methyltransferase Is Required for Stability of Other Polypeptides Involved in Coenzyme Q Biosynthesis*

Suzie W. Baba{ddagger}, Grigory I. Belogrudov{ddagger}, Justine C. Lee{ddagger}, Peter T. Lee{ddagger}, Jeff Strahan§, Jennifer N. Shepherd§, and Catherine F. Clarke{ddagger}

From the {ddagger}Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095 and the §Department of Chemistry, Gonzaga University, Spokane, Washington 99258

Received for publication, December 15, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Coenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquinone, or coenzyme Q (Q),1 is a polyprenylated benzoquinone lipid that is a critical component of the electron transport pathways of both eukaryotes and prokaryotes (1). Qn consists of a hydrophobic isoprenoid tail and a quinone head group. The tail length (n) varies depending on the organism studied; Saccharomyces cerevisiae contains Q6, Escherichia coli contains Q8, and humans contain Q10. The tail anchors Q in the membrane, while the head group is responsible for the redox chemistry, undergoing reversible redox cycling between the quinone (Q), semiquinone, and hydroquinone (QH2) forms. In eukaryotes Q is primarily associated with the inner mitochondrial membrane and is best known for its role in respiratory metabolism as a member of the electron transport chain shuttling electrons from Complex I (NADH:Q oxidoreductase) and Complex II (succinate:Q oxidoreductase) to Complex III (the cytochrome bc1 complex) (2). QH2 also acts as a lipid-soluble antioxidant, capable of scavenging lipid peroxyl radicals directly, or indirectly, by reducing {alpha}-tocopheroxyl radicals (3, 4). In the plasma membrane, Q participates in a trans-plasma membrane electron transport chain, in which intracellular NADH is oxidized and extracellular ascorbate free radicals are reduced (5). Recently, Q has been implicated in having a role in the rate of aging in the soil nematode Caenorhabditis elegans (6). Q supplementation is shown to be effective in slowing the progression of Parkinson's disease symptoms (7) and its effectiveness in treating Huntington's disease is being investigated (8).

The proposed biosynthetic pathway of Q was elucidated by the characterization of E. coli and S. cerevisiae Q-deficient mutants (9, 10). In yeast, Q-deficient mutant strains have been placed into eight complementation groups, coq1-coq8 (9, 11). They are respiratory deficient and hence unable to grow on nonfermentable carbon sources such as glycerol. The E. coli Q-less mutants sort into ten complementation groups, ubiA-ubiH, ubiX, and ispB, and fail to grow on media containing succinate as the sole carbon source (10). The C-methyltransferase step was identified as being defective in E. coli ubiE mutants which accumulate the Q biosynthetic intermediate DDMQH2 (Fig. 1, compound 3) (12, 13). The yeast COQ5 gene product was shown to be required for the C-methyltransferase step converting DDMQH2 to DMQH2 (Fig. 1, compounds 3 and 4, respectively) (14). The COQ5 and ubiE genes harbor four sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferase enzymes (13, 15). These motifs are found in a diverse group of enzymes that share a Class I methyltransferase structure (16).



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  		FIG. 1.
Methyltransferase steps in Q biosynthesis. In eukaryotes the C-methylation step requires the Coq5 polypeptide to convert 2-methoxy-6-polyprenyl-1,4-benzoquinol (compound 3) to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinol (compound 4). Coq3 catalyzes the O-methylation of 3,4-dihydroxy-5-polyprenyl benzoic acid (compound 1) to form 3-methoxy-4-hydroxy-5-farnesyl benzoic acid (compound 2), and also converts 2-farnesyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinol (demethyl-Q3 or compound 5) to Q3 (compound 6). The length of the polyisoprenoid tail is designated by n; S. cerevisiae, n = 6; human, n = 10; n = 3, farnesylated analogs used in this study.

 
A growing body of genetic evidence suggests that a complex of Coq polypeptides is responsible for Q biosynthesis. First, the same early intermediate, 3-hexaprenyl-4-hydroxybenzoate accumulates in the panel of Q-less yeast mutants (coq3, coq4, coq5, coq6, coq7, and coq8/abc1) (17). This is not the case with E. coli ubi mutants, where mutants accumulate the distinct Q-intermediate diagnostic of the blocked step of the Q biosynthetic pathway (18). Second, the coq7-1 point mutant (G104D), in contrast to the coq7-null mutant, accumulates DMQ, an intermediate only two steps away from Q (Fig. 1, compound 4) (19). Finally, levels of the Coq3 polypeptide and corresponding O-methyltransferase activity are substantially lower in each of the coq-null mutants (20). Taken together, these data suggest that a multisubunit complex of Coq polypeptides is involved in Q biosynthesis in yeast. This study identifies the Coq5 as a mitochondrial matrix polypeptide peripherally associated with the inner membrane, and demonstrates that Coq5p is essential for the stability and activity of other Coq polypeptides involved in Q biosynthesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Media—The S. cerevisiae strains used in this study are listed in Table I. Growth media for yeast (21) were prepared as described and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG (1% yeast extract, 2% peptone, 3% glycerol), and YPGal (1% yeast extract, 2% peptone, 2% galactose, 0.1% dextrose). E. coli were grown in Luria-Bertani (LB) broth. When required, ampicillin was added to a final concentration of 100 µg/ml. All solid media contained 2% Difco Bacto agar. Yeast and bacteria were grown at 30 °C and 37 °C, respectively.


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  		TABLE I
Genotypes and sources of S. cerevisiae strains

 
Construction of Yeast Expression Plasmids Containing E. coli ubiE— The pUE3 plasmid (13) contains the E. coli ubiE ORF fused to an amino-terminal mitochondrial leader sequence of COQ3, and expression is driven by the S. cerevisiae CYC1 promoter. pQM represents the single copy empty vector control for pUE3, and contains only the mitochondrial leader sequence and the CYC1 promoter segment (22). A 1.24-kb BamHI/KpnI fragment of pUE3 was inserted into the multicopy vector pRS426 (23), to generate pHUE3–1, a multicopy version of pUE3. To construct pS5PE1, pUE3 was digested with BamHI and EcoRI, to remove the DNA segment containing the CYC1 promoter, and a 1.0-kb fragment containing the promoter region of the COQ5 gene was inserted. The COQ5 promoter region (–1000 to –1) was prepared by PCR amplification of the template pBOB (14) with the primers pQ5P-1 (–1000 to –980; 5'-CGCGGATCCGCGTTGAAGGGATTCCTTTGAGG-3') and pQ5P-2 (–21 to –1; 5'-CCGGAATTCCGGTATATCTTTCTTGCTGCGAT-3'). The resulting 1.0-kb PCR product containing the COQ5 promoter was digested with BamHI and EcoRI, and this fragment replaced the CYC1 promoter in pUE3 as described. The 1.86-kb BamHI/KpnI fragment of pS5PE1 was inserted into pRS426 to generate pM5PE1, a multicopy version of pS5PE1.

Sequence Analysis—DNA segments containing the protein coding region, and 5'- and 3'-flanking regions of COQ5 alleles were produced by PCR amplification of genomic DNA from yeast strains CH83-B1 (coq5-2), CH256-3A (coq5-3), CH259-5D (coq5-4), and CH316-6B (coq5-5). The 5'-primer pBCOQ5 (5'-CCGTGATACTATCGGCGATA-3') corresponded to position –400 to –380, and the 3'-primer pTCOQ5 (5'-TGGCTATCACATGGCACAGG-3') corresponded to +1040 to +1020 relative to the +1 position of the COQ5 coding region. DNA sequence analysis of the PCR product was performed as described previously (13).

Generation of Antisera Against Coq5p and Western Blot Analysis—A segment of the COQ5 ORF (+91 to +924) encompassing the predicted mature Coq5p was amplified with the primers pHF5 (+91 to +113; 5'-GAAGATCTCAAAGAAGAAGAAGTTAATAGTC-3') and pHR5 (+924 to +896; 5'-GAAGATCTTTAAAVTTTAATGCCCCAATG-3'). An ~0.83-kb fragment of COQ5 ORF was digested with BglII and inserted into the BglII site of the expression vector pTrcHisB (Invitrogen) to generate pTHQ5–1. A fusion protein containing a His6 tag at the N terminus was overexpressed in the E. coli strain DH5{alpha}. Expression of the recombinant Coq5p was induced with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside at 37 °C and continued for 3 h. For purification of Coq5p, harvested cells were resuspended in 5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9 buffer containing 6 M urea and cells were disrupted by five freeze-thaw cycles and sonicated. The supernatant obtained after a 1-h centrifugation at 12,000 rpm was applied to a Ni(II)-NTA column (Qiagen). The column was washed with buffer (as above, but containing 20 mM imidazole), and the recombinant Coq5p was eluted with 250 mM imidazole in the above buffer containing 6 M urea. The His6-Coq5 fusion protein was further purified by 15% SDS-PAGE and transferred to an Immobilon P membrane. The electrophoretically pure preparation of the fusion protein was eluted from the membrane and used to generate antiserum in rabbits (Cocalico Biologicals, Inc., Reamstown, PA). The specificity of the antiserum was confirmed by immunoblotting against mitochondria from a wild type strain and a strain containing coq5::HIS3 disruption allele. Western blot analysis was carried out as described (24). The primary antibodies were used at the following dilutions: anti-Coq3p, 1:2000; anti-Coq5p, 1:2000; anti-{beta} subunit of F1-ATPase, 1:15,000; anti-Cyt c1, 1:1000; anti-Cyt b2, 1:5000; anti-CCPO, 1:5000; anti-Mge1p, 1:5000; and anti-porin, 1:5000. Horseradish peroxidase-linked secondary antibodies to rabbit IgG (Calbiochem) were used at a 1:1000 dilution.

Mitochondrial Localization of Coq5p—Yeast were grown in YPGal media to OD600 of ~4. Preparation of spheroplasts and cell lysate fractionation were performed as described (25). Crude mitochondria were further purified over a linear Nycodenz gradient (26). Proteinase K protection experiments were carried out as described in (27) using Nycodenz-purified mitochondria.

Mitochondrial subfractionation was carried out by generating mitoplasts (26) followed by sonication (3 x 30 s on ice slurry, 50% duty cycle, 4 output setting; Sonifier W350, Branson Sonic Power Co.). Mitochondrial membrane and soluble fractions were separated by centrifugation (150,000 x g, 60 min, 4 °C). The membrane fraction was washed by repeated centrifugation. Equal aliquots of starting mitochondria, intermembrane space, membrane, and matrix fractions were separated by 15% SDS-PAGE and after transfer to nitrocellulose were subjected to Western blot analysis.

In Vitro C-Methyltransferase Assay—Assays of C-methyltransferase activity were performed with 2-farnesyl-6-methoxy-1,4-benzoquinol (compound 3, Fig. 1) as substrate to a final concentration of 50 µM (14). The assay was modified so that reactions were similar to those described for the {gamma}-tocopherol methyltransferases (28). Each reaction mixture (500 µl) contained 50 mM Tricine-NaOH, pH 7.5, 1 mM MgCl2, 0.5 mM NADH, and 0.2–0.4 mg crude yeast mitochondrial protein. Reactions were started with the addition of S-adenosyl-L-[methyl-3H] methionine to a final concentration of 1 µM (PerkinElmer Life Science Products, 81.5 Ci/mmol) and incubated at 37 °C for 1 h. Assays performed under these conditions with wild-type mitochondria showed linear rates of product formation. Reactions were stopped by addition of 5 µl of glacial acetic acid and oxidized with 25 µl of fresh 1% ammonium cerium(IV) nitrate. Lipids were extracted with 2 ml of 1:2 (v/v) chloroform/methanol and 500 µl of 0.9% (w/v) NaCl and centrifuged at 2500 rpm for 5 min. The organic layer was transferred to a new tube and dried under nitrogen gas, separated by HPLC and assayed for radioactivity (22).

In Vitro O-Methyltransferase Assay—Protein concentration was determined with the BCA assay (Pierce) with bovine serum albumin as a standard. Assays of O-methyltransferase activity were performed with 3,4-dihydroxy-5-farnesylbenzoic acid (compound 1, Fig. 1) or 2-farnesyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinol (compound 5, Fig. 1) as substrate (29). The corresponding farnesylated analogs of methylated products (compounds 2 and 6, Fig. 1) served as the respective product standards. Each reaction mixture (250 µl) contained 50 mM sodium phosphate, pH 7.0, 1 mM ZnSO4, and 1 mg of crude yeast mitochondrial protein. Assays with compound 5 as substrate also contained 1 mM NADH. The final concentration of substrate per reaction was 50 µM. Reactions were started with the addition of S-adenosyl-L-[methyl-3H]methionine to a final concentration of 0.43 µM (PerkinElmer Life Science Products, 63.8 Ci/mmol) and incubated at 37 °C for 30 min. Reactions were stopped by addition of 5 µl glacial acetic acid. The lipids were extracted twice with 1 ml of 5:2 (v/v) hexane/ethanol, dried under nitrogen. HPLC and assays for radioactivity were performed as described above.

RNA Isolation and Northern Analysis—Total RNA was isolated from the yeast strains listed in Table I. Yeast were grown in YPGal media and harvested in mid-log phase (OD600 = 1.0). RNA was isolated by the hot phenol method as described (30). RNA was resolved by electrophoresis on a 1% formaldehyde gel and subsequently transferred to a Hybond-N+ charged nylon membrane (Amersham Biosciences). The membrane was rinsed in 2x SSC (1x SSC: 0.15 M sodium chloride, 0.014 M sodium citrate, pH 7.0), UV-treated, and prehybridized in hybridization buffer containing 7% SDS, 0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, and 1% bovine serum albumin, at 65 °C for 20 min. Hybridization was performed at 65 °C overnight in hybridization buffer containing both the CHC1 and one of the COQ radiolabeled probes. Membranes were washed once with 2x SSC, 0.1% SDS at 65 °C for 15 min and once with 0.2x SSC, 0.1% SDS at 65 °C for 15 min, and placed under a phosphorimager screen overnight and analyzed with a PhosphorImager (Molecular Dynamics, version 4.0).

Four radiolabeled probes were used. A DNA segment containing 440 bp of the COQ5 ORF (924 bp) was amplified from pRSHA5–1 by PCR with primers pHF5 and pSW5–2 (+507 to +482; 5'-CTTGAAATATTTTCCTTGTTCCAT-3'). A DNA segment containing the COQ3 ORF (951 bp) and 650 bp upstream was amplified from pRS12A2–2.5Sma (31) by PCR with primers pBC3–1 (–650 to –631; 5'-TAAATTTCTGAGCTCGCCCCCGGGTATTTCATTTG-3') and pBC3–2 (5'CGCGGGATCCATTCAGTCTCTGAATAGCCA-3') generating a product of 420 bp. A DNA segment containing the COQ4 ORF (1008 bp) was amplified from pTAP4 (COQ4)2 by PCR with primers pHF4 (+87 to +107; 5'-CGGGATCCGTACACCTTAGGCTCATTAAT-3') and pHR4 (+902 to +925; 5'-CGGGATCCTTAGATCCGGAGGAGTGTTA-3') generating a 851-bp product. A clathrin heavy chain specific probe was prepared from pCHCe200 (32) by PCR with primers pSWCHC1–1 (+120 to +149; 5'-GAATAAATACAAAGAGAATTAAGAAAAGTA-3') and pSWCHC1–2 (+892 to +865; 5'-GTTTTTGTTAGCCACAAAATTGATAAT-3'), generating a 672 bp clathrin (CHC1) DNA product. The resulting PCR products were separated on a 1% agarose gel and purified using a DNA purification kit (Qiagen). The purified PCR products were labeled by random priming with [{alpha}-32P]dCTP (3000 Ci/mmol, PerkinElmer Life Science Products) and the Prime-It RmT Random Primer Labeling Kit (Stratagene). Unincorporated nucleotides were eliminated with the NucTrap push column (Stratagene).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Subset of the Yeast coq5 Mutant Strains Are Rescued by E. coli ubiE—Previous work showed that a yeast strain harboring the coq5-2 mutation was rescued for growth on glycerol media by expressing E. coli ubiE, a functional homologue of COQ5 (14). The rescuing plasmid used in these studies, pUE3, contained the E. coli ubiE coding region fused in-frame to a mitochondrial leader sequence, and expression was driven by the yeast CYC1 promoter. To examine whether E. coli ubiE could completely substitute for yeast COQ5, a panel of coq5 mutants, including a coq5-null mutant (coq5{Delta}) were transformed with pUE3 and tested for the ability to grow on YPG media, which contains glycerol as a nonfermentable carbon source. As shown in Fig. 2, the E. coli ubiE gene restored growth of the yeast coq5-2 and coq5-5 mutants, but not of coq5{Delta}, coq5-3, or coq5-4 mutant yeast. These data indicate that the E. coli ubiE gene rescues only a subset of the yeast coq5 mutant strains. Identical results were obtained following transformation with pHUE3-1, a multicopy version of pUE3, and when ubiE was expressed from the yeast COQ5 promoter, present on either single copy (pS5PE1) or multicopy (pM5PE1) plasmids (Table II). Conversely, each of the coq5 mutants is rescued when transformed with plasmids containing the yeast COQ5 gene (Table II).



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  		FIG. 2.
Expression of E. coli ubiE restores growth of only a subset of the coq5 mutant yeast strains on media containing a non-fermentable carbon source. Yeast mutant strains (Table I) were transformed with plasmids containing either the S. cerevisiae COQ5 gene (COQ5), or the E. coli ubiE gene expressed from the yeast CYC1 promoter in frame with a mitochondrial leader sequence (pUE3).

 


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  		TABLE II
A subset of yeast coq5 mutant strains are rescued by E. coli ubiE

 
Complementation Analysis of coq5 Mutants—Complementation analysis was carried out with the yeast coq5 mutant strains. As shown in Table III, diploids resulting from the mating of coq5-3 and coq5-2, or coq5-3 and coq5-5 mutants, were able to grow on YPG media, while diploids produced in all other crosses were defective for growth on YPG. These results suggest that at least two categories of coq5 mutant alleles are present in the panel of coq5 mutants: one that is defective in the C-methyltransferase step (and can be rescued by E. coli ubiE), and another that is defective in a secondary function of Coq5p.


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  		TABLE III
Complementation of coq5-3 by only the coq5-2 and coq5-5 mutants

a and {alpha} coq5 mutant yeast strains were mated on YPD plates, and diploids were selected on the appropriate drop-out plate media and then replica plated on YPG, a non-fermentable carbon source. The (+) designates growth of the diploid on YPG plates and the (–) designates lack of growth on YPG plates.

 
Sequence Analysis of coq5 Mutant Alleles—To identify the defect in each of the coq5 mutants, DNA segments encompassing the COQ5 coding region plus 400 bp of 5'-flanking and 100 bp of 3'-flanking sequence were amplified from yeast strains bearing the coq5-2, coq5-3, coq5-4, or coq5-5 alleles. Sequence analysis revealed that each coq5 mutant allele harbored a unique nucleotide substitution, resulting in the following amino acid changes: G199D in coq5-2; G304D in coq5-3; W93STOP in coq5-4; and G121R in coq5-5 (Fig. 3). The early stop mutation present in coq5-4 results in a phenotype similar to the coq5{Delta} mutant. As with the coq5{Delta} mutant strain, coq5-4 is neither rescued by E. coli ubiE, nor does it complement the coq5-2 or coq5-5 mutants. The G199D mutation in coq5-2 affects an invariant glycine residue present in a conserved region adjacent to motif II, while the G121R mutation in coq5–5 affects a conserved glycine present in methyltransferase motif I. Both regions appear important for Coq5 C-methyltransferase activity (13) and it is likely that these amino acid substitutions would inactivate C-methyltransferase function. In contrast, the G304D mutation present in the coq5-3 polypeptide is located only four residues away from the C terminus. It seems less likely that C-methyltransferase activity of coq5-3 would be impaired, consistent with the interpretation of the complementation analysis, suggesting that the coq5-3 polypeptide retains C-methyltransferase activity. However, the coq5-3 mutation does result in a respiratory deficient phenotype, indicating that some other function of Coq5p is being compromised.



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  		FIG. 3.
Identification of the mutations in four different coq5 mutant strains. DNA segments containing the open reading frame, and 5'- and 3'-flanking sequences of the yeast COQ5 gene were amplified from genomic template DNA isolated from yeast strains CH83-B1 (coq5-2), CH256-3A (coq5-3), CH259-5D (coq5-4), and CH316-6B (coq5-5). Nucleotide sequences of the PCR products were determined. The following nucleotide changes were present in each of the four coq5 mutant alleles as well as in a wild-type parental yeast strain W303-1B: G65T (C22F), G124A (A42T), G189A, A441T, and G846A. The last three nucleotide changes did not change the amino acid, while the first two changes encoded amino acid substitutions at positions 22 and 42 as designated. For each of the coq5 mutant alleles, there was one unique nucleotide substitution. These mutations are designated by asterisks (*) on the figure as G596A (G199D) for coq5-2, G911A (G304D) for coq5-3, G279A (W93STOP) for coq5-4, and G361A (G121R) for coq5-5.

 
Assays of Methyltransferase Activity in Mitochondria Isolated from the coq5 Mutants—In vitro C-methyltransferase activity of Coq5p was assayed with the farnesylated analog of compound 3 (Fig. 1) and S-adenosyl-L-[methyl-3H]methionine. Mitochondria from wild-type yeast produced a radioactive product that co-migrated with the DMQ3 product standard on reverse phase HPLC (Fig. 4A; fraction 8). No activity was detected in mitochondria isolated from the coq5{Delta} strain. Assays were similarly performed with mitochondria isolated from the different coq5 mutant strains. Only the coq5-3 mutant had detectable C-methyltransferase activity (Fig. 4B). The amount of C-methyltransferase activity present in mitochondria isolated from the coq5-3 mutant was rather variable, ranging from 30 to 75% of wild type. A representative result is presented in Fig. 4B.



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  		FIG. 4.
Levels of C-methyltransferase activity in wild-type and coq5 mutant yeast strains. A, yeast mitochondria were prepared from W303-1A (WT) or W303{Delta}coq5 (delete) and in vitro C-methyltransferase assays were carried out with a farnesylated analog of compound 3 (Fig. 1) as the methyl-acceptor substrate and S-adenosyl-L-[methyl-3H]methionine. A lipid extract of the in vitro assay was separated by HPLC and the radioactivity contained in 1-min fractions was quantified. Radioactivity in fraction 8 co-eluted with the farnesylated product standard (compound 4, Fig. 1), and is depicted as (pmol of CH3 groups x mg protein–1 x h–1). B, assays were performed as described in panel A with mitochondria isolated from W303-1A (WT), W303{Delta}coq5 (delete), CH83-B3 (coq5-2), CH256-3A (coq5-3), CH259-5D (coq5-4), and CH316-6B (coq5-5) yeast strains. Radioactivity present in fraction 8 is expressed as pmol of CH3 groups x mg protein–1 x h–1. Data shown are representative of three independent experiments.

 
In vitro O-methyltransferase activity of Coq3p was assayed with the farnesylated analogs of either compound 1 or 5 (Fig. 1). Essentially no activity was detected in mitochondria isolated from coq5{Delta} yeast with either of the farnesylated substrate analogs (Fig. 5A), consistent with results obtained previously (20). Coq3 O-methyltransferase activity was readily detected in mitochondria isolated from coq5-2 and coq5-5 mutants regardless of substrate (Fig. 5, A and B). Previous assays have shown that assays of yeast Coq3 O-methyltransferase activity is much greater with the farnesylated analog of compound 5 as compared with compound 1 (29). Significantly, very low levels of O-methyltransferase activity were present in mitochondria isolated from either the coq5-3 or coq5-4 mutants (Fig. 5B). These data indicate that a subset of the coq5 mutants, coq5-2 and coq5-5, retain O-methyltransferase activity, while only the coq5-3 mutant retains C-methyltransferase activity.



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  		FIG. 5.
Levels of O-methyltransferase activity in the wild type and coq5 mutant yeast strains. Yeast mitochondria were prepared from W303-1A (WT), W303{Delta}coq5 (delete), CH83-B3 (coq5-2), CH256-3A (coq5-3), CH259-5D (coq5-4), and CH316-6B (coq5-5), and O-methyltransferase assays were carried out with farnesylated analogs of compound 1 or compound 5 as the methyl-acceptor substrate and S-adenosyl-L-[methyl-3H]methionine. A lipid extract of the in vitro assay was separated by HPLC and the radioactivity contained in 1-min fractions was quantified. Radioactivity co-eluting with the corresponding farnesylated product standard (compound 2) or (compound 6) is depicted as pmol of CH3 groups x mg protein–1 x h–1 in A and in an expanded graph in B. Error bars represent standard deviation from two methyltransferase assays of the same mitochondria sample, and data shown are representative of two independent experiments.

 
Submitochondrial Localization of Coq5 Polypeptide—To determine the submitochondrial localization of Coq5p, yeast mitochondria were further fractionated, and Coq5p was monitored with an antibody specific for yeast Coq5p. Purified mitochondria from wild-type yeast were subjected to treatment with hypotonic buffer, which disrupts the outer membrane while keeping the inner membrane intact, followed by sonication, releasing soluble matrix proteins into the supernatant after centrifugation. Western blot analysis of sub-mitochondrial fractions indicated that Coq5p remained associated with the membrane fractions, along with the integral membrane protein, cytochrome c1, while the soluble components of the intermembrane space, CCPO, or mitochondrial matrix, Mge1p, were released into the supernatant (Fig. 6A).



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  		FIG. 6.
Submitochondrial localization of Coq5p. All samples we separated by SDS-PAGE and analyzed by Western blotting with antiserum against Coq5p. Specific antisera recognizing mitochondrial marker proteins were used: Mge1, (Mge1) a soluble matrix protein;, cytochrome c peroxidase, (CCPO) and cytochrome b2, (Cytb2), both soluble proteins of the mitochondrial intermembrane space; and cytochrome c1, (Cytc1) an integral membrane protein of the mitochondrial inner membrane. A, Coq5p associates with a particulate fraction of mitochondria. Mitochondria (Mito) were subjected to a hypotonic shock to release intermembrane space proteins (IMS) and after sonication, separated into membrane (P) and soluble matrix protein (S) fractions. B, Coq5p is located on the matrix side of the inner mitochondrial membrane. Intact mitochondria, or mitoplasts, generated from mitochondria by a hypotonic treatment, were incubated with proteinase K (100 µg/ml) for 30 min on ice, in the presence or absence of 1% Triton X-100. C, Coq5p is extracted from the membrane with alkaline pH treatment. Mitochondria were treated with 0.1 M Na2CO3, pH 11.5, on ice for 30 min. Following centrifugation, equal aliquots (volumes) of soluble (S) and insoluble (P) fractions as well as total (T) mixture were analyzed.

 
To determine whether Coq5p associates with the outer or inner mitochondrial membrane, mitoplasts generated by hypotonic swelling of purified mitochondria were subjected to treatment with proteinase K in the presence and absence of the detergent Triton X-100. Coq5p was protected from proteolysis in intact mitochondria and mitoplasts, as was Mge1p, a soluble protein of the mitochondrial matrix, while cytochrome b2, a soluble intermembrane space polypeptide, was readily digested (Fig. 6B). Only a disruption of membrane structure with Triton X-100 rendered Coq5p protease sensitive. However, contrary to cytochrome c1, Coq5p is efficiently solubilized after alkaline treatment of whole mitochondria (Fig. 6C). These results indicate that Coq5p peripherally associates with the matrix side of the inner mitochondrial membrane.

Steady State Levels of Coq5, Coq3, and Coq4 Polypeptides— Steady state levels of Coq5 polypeptide were examined by Western blot analysis of mitochondria isolated from each of the coq5 mutant strains. Polypeptide levels are normal in coq5-2 and coq5-5 mutants, diminished in the coq5-3 mutant, and not detected in coq5{Delta} and coq5-4 mutants (Fig. 7). The low Coq5 polypeptide level seen in coq5-3 is curious, since significant levels of C-methyltransferase activity are detected.



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  		FIG. 7.
Western blot analysis of steady state levels of Coq5, Coq3, and Coq4 polypeptides in wild type and coq5 mutant yeast strains. Mitochondria were prepared from wild type and coq5 mutant yeast strains. Equal aliquots of protein (crude mitochondria; 13 µg) were separated by SDS-PAGE and analyzed by Western blotting with antibodies against Coq5, Coq3, and Coq4 polypeptides. As a control for steady state level of other yeast mitochondria constituents, specific antisera were used against marker proteins including Mge1p, {beta}F1 (a peripheral protein of the inner membrane on the matrix side), Cyt c1, CCPO, and Porin, (an outer membrane protein).

 
The steady state level of Coq3p has previously been shown to be greatly diminished in the coq5{Delta} mutant yeast (20). Western blot analysis with antibodies specific for yeast Coq3p or Coq4p revealed a loss of these two polypeptides in coq5{Delta}, coq5-3, and coq5-4 mutant yeast (Fig. 7). The similar behavior of the steady state levels of Coq3, Coq4, and Coq5 suggests that these polypeptides may be part of a complex.

Steady State mRNA Levels of COQ5, COQ3, and COQ4 —To determine whether levels of COQ5, COQ4, or COQ3 RNAs are altered in the panel of coq5 mutants, Northern blot analysis was performed. Steady state COQ5 RNA levels are not detected in coq5{Delta} and coq5-4 mutants (Fig. 8). It is likely that the absence of stable mRNA in coq5-4 mutant may be attributed to nonsense-mediated mRNA decay (33). When normalized to the amount of the CHC1 signal, steady state COQ5 mRNA levels in the coq5-2, coq5-3, and coq5-5 mutants are very similar to wild type. These data indicate that the level of Coq5p is not simply reflecting the abundance of COQ5 RNA, since the level of coq5-3 RNA is not decreased relative to coq5-2 RNA. Similarly, the amount of COQ3 and COQ4 steady state mRNA levels, when normalized for the amount of the CHC1 signal, do not account for the decreased levels of Coq3p and Coq4p in the coq5-3 mutant strain (Fig. 8).



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  		FIG. 8.
COQ5, COQ3, and COQ4 steady state mRNA levels. Yeast total RNA was prepared and analyzed as described under "Experimental Procedures." Equal aliquots (10 µg) were loaded in each lane of a denaturing agarose gel and blotted to a charged nylon membrane. Hybridization was performed with 32P-labeled probes corresponding to DNA segments of COQ5, COQ3, or COQ4 yeast genes. A 32P-labeled probe corresponding to the yeast clathrin heavy chain (CHC1) gene was used as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Coq5 and its E. coli homolog, ubiE, are C-methyltransferases required for the conversion of compound 3 to compound 4 in coenzyme Q biosynthesis (Fig. 1). Yeast coq5 mutants are respiratory defective, lack Q, and are unable to grow on glycerol, while E. coli ubiE mutants lack Q, accumulate compound 3, and are unable to grow on succinate. Previous work showed interspecific complementation of Coq5 and ubiE suggesting that they are functional homologs (14).

The genetic studies reported in the current work show that although the S. cerevisiae COQ5 gene rescued each of the coq5 mutants, only the coq5-2 and coq5-5 mutant strains were rescued by expression of the E. coli ubiE gene. Sequence analysis revealed that each coq5 mutant allele harbors a unique nucleotide substitution. Neither full-length Coq5p, nor a smaller fragment was detected in the coq5-4 mutant (W93STOP), accounting for its similar phenotype to that of the coq5 deletion mutant (coq5{Delta}). The coq5-5 (G121R) and coq5-2 (G199D) mutants are likely to be defective in C-methyltransferase activity since the mutation in each lies in or near a methyltransferase motif, respectively. Indeed, direct measurements of C-methyltransferase activity in mitochondria prepared from our panel of coq5 mutant strains revealed that only coq5-3 mitochondria exhibited an appreciable amount of C-methyltransferase activity (Fig. 4). Interestingly, the amino acid substitution in coq5-2 recapitulates that found in the ubiE401 mutant (G142D) and affects a conserved glycine residue thought to be critical for substrate binding (13). The amino acid substitution present in the Coq5-3 polypeptide (G304D) is located four amino acids away from the C terminus. The phenotype of this mutant is very interesting; in mating studies, the coq5-3 mutant strain complements the coq5-2 and coq5-5 mutants for growth on glycerol, yet the coq5-3 mutant is respiratory deficient on its own.

Two O-methyltranferase steps occur in coenzyme Q biosynthesis and Coq3p catalyzes both. In coq5-3, coq5-4 and coq5{Delta} mutants, O-methyltransferase activity is drastically reduced (Fig. 5), in agreement with Western blot analysis showing a loss of Coq3 polypeptide in these two mutants (Fig. 7). Interestingly, a similar loss of Coq4 polypeptide is observed in the coq5-3, coq5-4, and coq5{Delta} mutants (Fig. 7). A low level of Coq5 polypeptide is detected in the coq5-3 mutant, and this mutant is able to grow on YPG after 10 days at 30 °C, suggesting the presence of a low level of C-methyltransferase activity. This activity was indeed detected in the C-methyltransferase assay of mitochondria isolated from the coq5-3 mutant strain. However, this low level of Coq5 polypeptide appears to be insufficient for detectable Coq3 activity (Fig. 5) or Coq3 or Coq4 polypeptide stability (Fig. 7). Coq3, Coq4, and Coq5 polypeptide levels are comparable to wild type in only the coq5-2 and coq5-5 mutants. These data indicate that stable, but catalytically inactive Coq5p, is sufficient for the expression of stable and active Coq3p. Thus, at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis, and second, it is involved in stabilizing Coq3p and Coq4p required for coenzyme Q biosynthesis. The stabilizing effect of Coq5p may be mediated by the C terminus of the polypeptide, since the glycine affected by the mutation coq5-3 is highly conserved (Fig. 3).

To better understand the level of regulation of COQ5 gene expression, Northern blot analysis was carried out on total RNA isolated from the yeast coq5 mutant panel. The COQ5 promoter contains consensus sites for Mig1, Rtg1/2/3, and Hap2/3/4; these factors are involved in regulating COQ5 expression depending on carbon source (34). In the present study we have found the level of COQ5 RNA was not decreased in the coq5-3 mutant. Thus the decrease in Coq5-3p must be due to post-transcriptional control. Such post-transcriptional control must also account for the decreased levels of Coq3p and Coq4p in the coq5-3, coq5-4, and coq5{Delta} mutants, as steady state levels of COQ3 and COQ4 mRNAs show little change in response to the type of coq5 mutation (Fig. 8).

In agreement with previous work (14, 35), Western blot analysis of the yeast subcellular fractions with antiserum against Coq5p has revealed association of the polypeptide with the mitochondria (data not shown). Detailed localization of Coq5p (Fig. 6) indicates that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Previously, Dibrov et al. (35) employed a fusion of c-Myc epitope attached to the C terminus of Coq5p to demonstrate the predominant localization of the fusion polypeptide in the mitochondrial matrix. The discrepancy in characterizing Coq5p as a peripheral membrane protein (current study) or soluble matrix protein (35) may be due to perturbation in the interaction of Coq5p with the inner mitochondrial membrane following either addition of the c-Myc epitope to the C terminus of the polypeptide or after treating mitoplasts with 2 M urea which may be sufficient to displace Coq5p from the inner membrane. Indeed, the role of the C terminus of Coq5p either in polypeptide folding or protein-protein/protein-membrane interactions is further emphasized by the fact that the steady state level of Coq5p in mitochondria of coq5-3 mutant is greatly diminished (Fig. 7).

A complex of Coq polypeptides is thought to be involved in Q biosynthesis based on a number of findings. First, the same predominant intermediate in the Q biosynthetic pathway accumulates in yeast strains harboring a deletion in coq3, coq4, coq5, coq6, coq7, or abc1/coq8 (17) while a coq7-1 point mutant (G104D) accumulates the Q intermediate predicted to be the substrate of the Coq7 polypeptide (19, 36). The requirement of stable Coq5 polypeptide for Coq3 polypeptide stability and O-methyltransferase activity suggests that Coq5p and Coq3p may reside together in a complex. Also, yeast Coq3p requires other COQ gene products for both O-methyltransferase activity and for high steady state polypeptide levels (20). Yeast two-hybrid screens have shown Coq5p to interact with Snf4, a protein kinase activator in the cytoplasm and nucleus of S. cerevisiae (37). However, mitochondrial protein partners for Coq5p have not yet been found. Our future studies are aimed at identifying the partner proteins that interact with Coq5p and Coq3p and assessing the structural organization of other COQ gene products in a multisubunit Q biosynthetic complex.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM45952 (to C. F. C.), and National Science Foundation Grants CHE-0077972 and CHE-0135091 (to J. N. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569. Tel.: 310-825-0771; Fax: 310-206-5213; E-mail: cathy{at}mbi.ucla.edu.

1 The abbreviations used are: Q, coenzyme Q (ubiquinone); QH2, ubiquinol; DDMQH2, demethyl demethoxy ubiquinol; DDMQ, demethyl demethoxy ubiquinone or 2-methoxy-6-polyprenyl-1,4-benzoquinone; DMQH2, demethoxy ubiquinol; DMQ, demethoxy ubiquinone or 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone; DHFB, dihydroxy farnesyl benzoate or 3,4-dihydroxy-5-polyprenyl benzoic acid; DMeQ3, demethyl Q3 or 3-methoxy-4-hydroxy-5-farnesyl benzoic acid; HPLC, high-performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. Back

2 M. Gulmezian and C. F. Clarke, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank the members of the Clarke laboratory for helpful comments and discussions. We thank Dr. A. Tzagoloff for providing the original coq5 mutant strains and Drs. M. Yaffe, G. Schatz, C. Koehler, and W. Neupert for the generous gifts of antisera.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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