The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNA Gln to glutaminyl-tRNA Gln . Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtf1 mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F 1 -F 0 ATP synthase that is degraded, thereby preventing assembly of the F 0 sector. Allotopic expression of recoded ATP8 and COX2 did not significantly improve growth of gtf1 mutants on respiratory substrates. However, ts gft1 mutants are partially rescued by overexpression of PET112 and HER2 that code for the yeast homologues of the catalytic subunits of bacterial AdT. Additionally, B66, a her2 point mutant has a phenotype similar to that of gtf1 mutants. These results provide genetic support for the essentiality, in vivo, of the GatF subunit of the heterotrimeric AdT that catalyzes formation of glutaminyl-tRNA
Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.
Coenzyme Q (CoQ) is a molecule of essential function in the transfer of electrons of the mitochondrial respiratory chain. In Saccharomyces cerevisiae, CoQ is constituted by a benzene ring associated with a polyprenyl chain with 6 repetition units, being therefore also denominated CoQ6 or Q6. Thirteen nuclear genes have already been identified (COQ1-COQ11, ARH1 and YAH1) required for coenzyme Q biosynthesis. Most of Coq products are physically associated in a biosynthetic complex anchored at the mitochondrial internal membrane. In this project, we identified Coq3p and Coq7p residues relevant for their respective role in CoQ synthesis combining bioinformatics analyzes with phenotypic tests for functional mapping. Coq7p is a carboxylate-bridged di-iron protein that catalyzes the hydroxylation of demetoxy-Q6 (DMQ6), the last monooxygenase step in the synthesis of CoQ. In this study, we found a group of residues that modulate the activity and stability of Coq7p: D53, R57, V111 and S114. While R57, V111 and S114 are highly conserved residues, V111 and S114 are correlated in communities of coevolution. We also demonstrate that the double mutant S114A, V111G and the mutant S114E have respiratory deficiency at non-permissive temperature, in addition to accumulating of the intermediate DMQ6 and low amounts of Q6, thus concluding that phosmimetic S114E inhibits the activity of Coq7p. Hence, we propose that the phosphorylation of S114 is required to move a loop between helices 2 and 3, thus affecting the activity of the catalytic center Coq7p. For its part, Coq3p acts as a methyltransferase, catalyzing different steps during biosynthesis of CoQ. Here we identified residues that collaborate for functional activity of Coq3p: E123,
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