The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis

Abstract

Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E(121)K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.

Fig. 1

The Coq4p sequence contains a catalytic zinc ligand motif conserved in prokaryotic species. Current database searching of prokaryotic databases reveals Coq4p homologs among alpha-, beta-, delta-, and gamma-proteobacterial classes, as well as representatives within cyanobacteria. These homologues are identified as members of COG 5031.1 (pfam05019). The Gene bank identifier (gi) numbers for the protein sequences shown, in order top to bottom are: 24212654, 75701042, 85375819, 72122094, 108762091, and 119459668. The asterisks indicate the G₁₂₀E, E₁₂₁K, and E₂₂₆K substitutions present in the coq4-3, coq4-2, and coq4-1 alleles, respectively. The boxed residues designate the location of a putative catalytic zinc ligand motif.

Fig. 2

Coq3p, Coq4p and other Coq polypeptides are stable in mitochondria isolated from coq4 point mutants. Panel A; Mitochondria (75 μg protein) were analyzed by SDS-PAGE, and immunoblotted with anti-sera to Coq4p. Panel B, Purified mitochondria (10 μg protein) from the designated yeast strains were subjected to similar SDS-PAGE and blotting conditions. The gels were transferred to polyvinylidene fluoride membranes and standard immunodetection with the indicated antisera to Coq3, Coq4, Coq7 or Rip1 polypeptides was performed. Images were obtained via Bio-Rad Fx imager using VersaDoc^™software.

Fig. 3

Yeast coq4 point mutants have defects in Q biosynthesis. Lipid extracts prepared from the designated yeast strains labeled with [U-¹⁴C]4-hydroxybenzoic acid were separated by reverse phase HPLC, 1 ml fractions collected, and ¹⁴C-radioactivity was determined by scintillation counting. Values are plotted as the ¹⁴C-radioactivity in dpm. A coenzyme Q₆ standard eluted in fraction 17.

Fig. 4

Coq3, Coq4, and Coq7 are displaced from high molecular mass complex in coq4-1 mutant mitochondria as compared to wild-type. A, Digitonin-extracts (2:1, g/g, detergent/protein) of mitochondria (2 mg protein) from wild-type W303 (white bars) versus coq4-1 (black bars) were separated by Superose 6 gel filtration chromatography. An aliquot of each fraction (200 of 600 μl) was assayed for Coq3-dependent O-methyltransferase activity as described in Experimental Procedures, and is depicted as pmol methyl groups/fraction/h. B, Coq3, Coq4, Coq7 or Rip1 polypeptides were detected in eluate fractions by immunoblot analyses. The elution positions of Coq3p, Coq4p and Coq7p in wild-type mitochondria are as determined previously [17], and are shown for purposes of comparison. The column void volume was established by the elution position of the vault particles in fraction 13. Elution position of calibration standards are indicated: 669 kDa, thyroglobulin; 440 kDa, ferritin; and 67 kDa, bovine serum albumin.

Fig. 5

Coq4p is displaced from high molecular mass in the coq4-1 mutant strain. Upper panel: 200 μg of mitochondria were isolated from wild-type W303-1A, solubilized with digitonin as described in Figure 4, and subjected to BN-PAGE (5–13.5% gradient gel) in the first dimension and SDS-PAGE (10–13%) in the second dimension, followed by immunoblot analysis with antisera to Coq4. Aliquots of control mitochondria from a coq4 null mutant (Δ4) or from W303-1A (wt) were subjected to just the second dimension (SDS-PAGE) in lanes adjacent to the BN-gel separation (upper panel only). Lower panel: 200 μg of mitochondria were isolated from the C9E1 coq4-1 mutant. In both panels, the asterisks designate the Coq4p signal, (distinct from the non-specific signal identified in the coq4 null mutant – see upper panel). The separation of protein markers in the first dimension is indicated: 669kDa, thyroglobulin; 440 kDa, ferritin; 232 kDa, catalase; 140 kDa, lactate dehydrogenase, and 67 kDa, bovine serum albumin.

Fig. 6

Coq3p has a high mass location in wild-type mitochondria and is displaced in mitochondria from coq3, atp2, coq4-1, and coq4-2 mutant yeast strains. One-dimensional BN-PAGE analyses (5–13.5% gradient gel) of digitonin-solubilized mitochondria from coq3 null (Δcoq3), wild-type (W303), coq4-1 or coq4-2 yeast strains. A, immuno-detection of Coq3p. B, the blot in A was stripped and re-probed with antisera to Rip1p.