Mutations in the Yeast MRF1 Gene Encoding Mitochondrial Release Factor Inhibit Translation on Mitochondrial Ribosomes

Joanna Towpik,Agnieszka Chaciñska,Malgorzata Cieśla,Krzysztof Ginalski,Magdalena Boguta

doi:10.1074/jbc.m312856200

Abstract

Although the control of mitochondrial translation in the yeast Saccharomyces cerevisiae has been studied extensively, the mechanism of termination remains obscure. Ten mutations isolated in a genetic screen for read-through of premature stop codons in mitochondrial genes were localized in the chromosomal gene encoding the mitochondrial release factor mRF1. The mrf1-13 and mrf1-780 mutant genes, in contrast to other alleles, caused a non-respiratory phenotype that correlated with decreased expression of mitochondrial genes as well as a reporter ARG8(m) gene inserted into mitochondrial DNA. The steady-state levels of several mitochondrially encoded proteins, but not their mRNAs, were dramatically decreased in mrf1-13 and mrf1-780 cells. Structural models of mRF1 were constructed, allowing localization of residues substituted in the mrf1 mutants and offering an insight into the possible mechanism by which these mutations change the mitochondrial translation termination fidelity. Inhibition of mitochondrial translation in mrf1-13 and mrf1-780 correlated with the three-dimensional localization of the mutated residues close to the PST motif presumably involved in the recognition of stop codons in mitochondrial mRNA.

Highlights

The control of mitochondrial translation in the yeast Saccharomyces cerevisiae has been studied extensively, the mechanism of termination remains obscure
Ten mutations isolated in a genetic screen for read-through of premature stop codons in mitochondrial genes were localized in the chromosomal gene encoding the mitochondrial release factor mRF1
The mrf1–13 and mrf1–780 mutant genes, in contrast to other alleles, caused a non-respiratory phenotype that correlated with decreased expression of mitochondrial genes as well as a reporter ARG8m gene inserted into mitochondrial DNA

Summary

EXPERIMENTAL PROCEDURES

Media and Growth Conditions—Rich media contained YPD (1% yeast extract, 2% peptone, and 2% glucose) and 2% galactose (YPGal) or. Homologous recombination, forced by growth of the integrant on SC medium supplemented with 5-fluoroorotic acid (2 mg/ml), resulted in excision of the URA3 plasmid and loss of either the MRF1 or mrf allele. Cytoduction was applied to transfer mitochondria containing the ARG8m reporter gene from donor cells (DFS189) into the nuclear mrf 780 background of recipient cell MJ14-1D [rho0] harboring the YCp314MRF1 plasmid. The presence of the ARG8m mitochondrial reporter gene was confirmed by a cross with the tester strain AK5-13B (recombination of mtDNA carrying the point cox2-V25 mitϪ mutation with mtDNA carrying the cox3::ARG8m deletion gives wild type mtDNA, leading to the recovery of the Glyϩ phenotype). In Vivo Labeling of Mitochondrial Translation Products—Cells grown as described above were suspended in buffer P (40 mM KPi, pH 7.0, 0.45% glucose, 0.0077% Complete supplement mixture-methionine (BIO101)) and incubated on a shaker for 30 min at 30 °C. A C␣ trace model of mRF1 bound to the ribosome was derived using the low resolution structure of E. coli RF2 in a complex with the ribosome [26]

RESULTS

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DISCUSSION