Functional characterization of human COQ4, a gene required for Coenzyme Q10 biosynthesis

Abstract

Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy. The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis. The human transcript is detected in Northern blots as a approximately 1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333-23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4(null) yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ(10) deficiency.

Fig. 1

Structure and expression pattern of human COQ4, and alignment of Coq4p of different species. (A) Human Coq4p aligned with Coq4 proteins from different eukaryotic species. Conserved residues are boxed. (B) COQ4 expression in different tissues. Radiolabeled probes (see text) were hybridized to a commercial preblotted membrane (FirstChoice Human Blot 1 membrane-Ambion Inc., Austin TX, USA) containing 2 μg/lane of poly(A)+ RNA from 10 human tissues. Radioactivity was detected with a Storm PhosphoImager (Molecular Dynamics, Sunnyvale, CA, USA) after an overnight exposure. (C) Structure of the COQ4 5′ region with the different transcription initiation sites detected by RACE experiments. The percentages indicate the relative abundance of each individual transcript (a total of 100 colonies were analyzed).

Fig. 2

Subcellular localization of the human COQ4 gene products. HeLa cells stably expressing mtRFP were transiently transfected with plasmids expressing COQ4-Isoform 1-GFP (A–C), COQ4-Isoform 2-GFP and GFP (D–F), or native GFP (G–I), and visualized using a Nikon Video Confocal Microscope.

Fig. 3

The human COQ4 gene restores the growth in non-fermentable substrates and coenzyme Q synthesis in COQ4^null yeast. (A) Cells grown in SDc-ura 2% galactose were inoculated into YPG agar plates. The initial suspension at 0.5 U OD 660 nm/mL was diluted 1/10 twice. Three microliters of cells solutions was spotted onto YPG plates which were incubated at 30 °C for three days. (B) The same SDc-ura 2% galactose cultures at 0.1 U OD 660 nm/mL were inoculated into liquid YPG medium. Growth was monitored measuring the OD at 660 nm. Data correspond to the average of three measures. The experiment is representative of a set of three. (C) Mitochondrial samples from wild-type cells (BY4741) and COQ4 mutant cells harboring the human COQ4 gene (∆COQ4:hCOQ4) and the yeast COQ4 gene (∆COQ4:yCOQ4) grown in YPG were subjected to lipid extraction and HPLC-ECD quinone separation and quantification as reported in the text. Data corresponds to the average ± SD of at least three determinations.