Phenotypic suppression and nuclear accommodation of the mit ? oxi1-V25 mutation in isolated yeast mitochondria

Abstract

Phenotypic suppression by the antibiotic, paromomycin, of the mitochondrial oxi1- -V25 mutation, a mutation which arrests by premature ochre codon the synthesis of the cox II subunit, was studied in isolated yeast mitochondria competent in translation. This antibiotic is known to suppress the mutation in vivo (Dujardin et al. 1984) and allowed in vitro, at concentrations of 20-1100 micrograms per ml. the synthesis of the cox II subunit. This strongly suggests that phenotypic suppression of mit- mutations is due to the direct action of paromomycin on mitochondrial ribosomes. The effect of paromomycin bears a resemblance to the function of the omnipotent nuclear suppressor mutation R705. The nuclear suppression was expressed in isolated mitochondria; suppressor mutation influenced the structure of the mitoribosome. Therefore, it appears that mitoribosomes are indeed the common target in the phenotypical and genetic nuclear suppression of the oxi1-V25 mutation.

Translational readthrough of a termination codon in the yeast mitochondrial mRNA VAR1 as a result of mutation in the release factor mRF1.

“…To confirm that the appearance of V2p is really the result of an alteration in translation, we assessed whether its synthesis is affected by paromomycin, an antibiotic that changes translation fidelity of cytosolic as well as of mitochondrial ribosomes (Wilhelm et al, 1978;Dujardin et al, 1984;Zagorski et al, 1987a). Translation in the presence of Na 2 [ 35 S]O 4 was monitored in mitochondria incubated either with or without paromomycin (see Materials and Methods) after isolation from the wild type, mitas well as from the mrf1-136 and mrf1-145 cells.…”

“…Translation in isolated mitochondria. Isolation of mitochondria and labeling of their translation products with Na 2 [ 35 S]O 4 were based on the method of McKee and Poyton (1984) with the modifications proposed by Zagorski et al (1987a) and Kozlowski & Zagorski (1988). Paromomycin was added to the translation mixtures at 400 mg ml -1 .…”