Characterization of the COQ5 Gene from Saccharomyces cerevisiae EVIDENCE FOR A C-METHYLTRANSFERASE IN UBIQUINONE BIOSYNTHESIS

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 (GenBank TM accession number Z49210) 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.

Phenotypes of Fission Yeast Defective in Ubiquinone Production Due to Disruption of the Gene for p -Hydroxybenzoate Polyprenyl Diphosphate Transferase

“…Since ubiquinone biosynthetic enzymes are localized to the mitochondria of S. cerevisiae (4,11,27), it has been suggested that ubiquinone biosynthesis occurs in the mitochondria. To assess the case for homologous enzymes from S. pombe, the localization of S. pombe Ppt1 was examined by Ppt1-green fluorescent protein FIG.…”

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

“…Medium was prepared as described (31) and included YPD (2% glucose, 1% yeast extract, 2% peptone), YPgal (2% galactose, 0.1% glucose, 1% yeast extract, 2% peptone), and YPG (3% glycerol, 1% yeast extract, 2% peptone). Synthetic dextrose/minimal medium was prepared as described (32) and consisted of all components (SDcomplete) or all components minus histidine (SDϪHis). Drop out dextrose medium (DOD) was prepared as described (33), except that galactose was replaced with 2% dextrose.…”

A Defect in Coenzyme Q Biosynthesis Is Responsible for the Respiratory Deficiency in Saccharomyces cerevisiae abc1Mutants

“…Growth media for yeast were prepared as described (34) and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPE (1% yeast extract, 2% peptone, 2% ethanol), YPGal (1% yeast extract, 2% peptone, 2% galactose), YPG (1% yeast extract, 2% peptone, 3% glycerol), SDC (0.18% yeast nitrogen base without amino acids, 2% dextrose, 0.14% NaH 2 PO 4 , 0.5% (NH 4 ) 2 SO 4 , complete amino acid supplement), SD-Leu (SDC minus leucine), and SD-Ura (SDC minus uracil). The complete supplement was modified so that the final concentration of each component was as described (23). Semisynthetic lactate media was prepared as described (35).…”

“…Coq3p is located in the mitochondrial matrix, peripherally associated with the inner membrane and catalyzes both O-methylation steps in Q biosynthesis (22). Coq5p functions as a C-methyltransferase in the mitochondria (23,24). Coq6p is likely to function as a flavin-dependent monooxygenase, adding one or more of the ring oxygens (20).…”