Abstract
Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. Q-deficient mutants of Saccharomyces cerevisiae fall into eight complementation groups (coq1-coq8). Yeast mutants from the coq5 complementation group lack Q and as a result are respiration-defective and fail to grow on nonfermentable carbon sources. A nuclear gene, designated COQ5 was isolated from a yeast genomic library based on its ability to restore growth of a representative coq5 mutant on media containing glycerol as the sole carbon source. The DNA segment responsible for the complementation contained an open reading frame (GenBankTM accession number Z49210Z49210) with 44% sequence identity over 262 amino acids to UbiE, which is required for a C-methyltransferase step in the Q and menaquinone biosynthetic pathways in Escherichia coli. Both the ubiE and COQ5 coding sequences contain sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. A gene fusion expressing a biotinylated form of Coq5p retains function, as assayed by the complementation of the coq5 mutant. This Coq5-biotinylated fusion protein is located in mitochondria. The synthesis of two farnesylated analogs of intermediates in the ubiquinone biosynthetic pathway is reported. These reagents have been used to develop in vitro C-methylation assays with isolated yeast mitochondria. These studies show that Coq5p is required for the C-methyltransferase step that converts 2-methoxy-6-polyprenyl-1, 4-benzoquinone to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone.
Highlights
Ubiquinone is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes
Cloning and Sequencing of the COQ5 Gene—The coq5 complementation group (G17) is composed of 20 independent isolates that contain recessive nuclear mutations resulting in the loss of respiration and Q synthesis [19]
The amino acid sequence of the Coq5 polypeptide is 44% identical over 262 amino acids with the E. coli UbiE polypeptide, which is required for the C-methylation reaction in Q biosynthesis [25]
Summary
Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. The primary function of Q is to transport electrons from Complex I or II to the cytochrome bc complex in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes [1] This cycle is carried out by a series of reductions and oxidations of the head group of Q. Yeast coq mutant strains (representative mutants from coq3– coq groups) when grown in the presence of p-[U-14C]hydroxybenzoic acid, a biosynthetic precursor of Q, fail to produce Q and accumulate an early intermediate that corresponds to 3-hexaprenyl-4-hydroxybenzoic acid [21] The presence of this intermediate is not necessarily diagnostic of the affected biosynthetic step, since it accumulates in wild-type yeast [21, 22]. The current study employs the strategy of comparing both sequence and function of the yeast COQ and E. coli ubi gene products to study the C-methylation step in yeast Q biosynthe-
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