Vol. 52 No. 1/2005 129–137 QUARTERLY

Yeast mitochondrial DNA codes for eight major polypeptides. Translation of he mitochondrially encoded polypeptides in strains with mutated mitochondrial release factor, mRF1, was found to result in the synthesis of a novel protein, V2. Different mrf1 alleles were associated with different efficiency of V2p synthesis. Translation of V2p was enhanced by paromomycin. Comparative analysis of peptides resulting from protease digestion indicated that V2p is a derivative of Var1p. According to our hypothesis, V2p represents a readthrough product of the natural stop codon in VAR1 mRNA.

could be restored by nuclear mutations, thus providing phenotypes amenable to fast and simple screening.A systematic search for nuclear suppressors of mit -resulted in the identification of several chromosomal genes that have a well-defined impact on mitochondrial functions (Herbert et al., 1988;Asher et al., 1989;Altamura et al., 1992;Boguta et al., 1992;Ekwall et al., 1992).
We have studied suppressors of mit -mutants that act at the level of mitochondrial translation.Two nuclear mutants, nam3-1 and nam3-2, carrying a suppressor of the cob-M2001 mit -ochre mutation localized in the maturase coding region and preventing splicing of intron bi2 in the mitochondrial COB gene, were isolated (Kruszewska & Slonimski, 1984a).Genetic studies indicated an allele-specific, gene non-specific omnipotent mode of suppression that could result from decreased translation fidelity of premature stop codons (Kruszewska & Slonimski, 1984b;Zagorski et al., 1987b).This hypothesis was in part verified by direct biochemical data (Boguta et al., 1988;Mieszczak & Zagorski, 1987b).Recent studies identified nam3-1 and nam3-2 as alleles of the nuclear gene MRF1 encoding the mitochondrial release factor corresponding, respectively, to mrf1-145 352 S®I and mrf1-136 216 S®Y (Towpik et al., 2004).Construction of a structural model of the mRF1 molecule allowed for localization of the substituted serines, 352 S and 216 S, in the vicinity of the codon recognition motif.This localization confirms the genetic data indicating the effect of mrf1-136 and mrf1-145 on the fidelity of termination.
Here we analyse precisely the proteins synthesized in mrf1-136 and mrf1-145 mitochondria, searching for polypeptides possibly representing the products of reading through the natural stop codons.We observed the synthesis of a novel protein, V2, which is an extended form of Var1p.Thus, mutations in the mRF1 encoding gene allow mitoribosomes to misread a premature as well as a natural stop codon.

Strains, media and growth conditions.
The yeast strains used in these studies, derived from P.P. Slonimski's collection, are listed in Table 1.
Media used for yeast growth were YPD (2% glucose, 2% peptone, 1% yeast extract) or YPGly (2% glycerol, 2% peptone, 1% yeast extract).The growth rate in liquid cultures at 30°C was monitored by A 600 measurements.For isolation of mitochondria and in vivo labeling, yeast cells were harvested in the mid exponential phase of growth.
In vivo labeling of mitochondrial translation products.dium and incubated for 2 h at 30°C on a shaker before the addition of cycloheximide to a final concentration of 600 mg ml -1 .After 4 min 0.5 mCi/ml Na 2 [ 35 S]O 4 was added and samples were collected after 1 h.The labeling reaction was terminated by chilling the cells on ice and simultaneous addition of unlabeled sulphate.After centrifugation the cells were resuspended in a buffer containing 250 mM mannitol, 20 mM Tris/HCl, pH 7.5, 1 mM EDTA and 1 mM phenylmethylsulphonyl fluoride (PMSF).Mitochondria were isolated as described by Claisse et al. (1980).Samples of mitochondrial proteins (50 000-200 000 c.p.m.each) were analysed by SDS/PAGE on gel slabs containing an exponential gradient (10-15%) of polyacrylamide.The gels were fixed, dried, and exposed to Kodak X-ray films, as described by Claisse et al. (1977).
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 .Analysis by SDS/PAGE was as described above.
Peptide maps.Peptide maps were based on the method of Cleveland et al. (1977) with the modifications described by Claisse et al. (1980).Selected labeled bands were cut out from dried SDS/PAGE slabs.Proteins were treated with S. aureus V8 protease (Miles Laboratories) using, respectively, 0.1 mg/sample or 50 mg/sample for limited or extensive digestion and the peptides were analysed by 15% SDS/PAGE.For double digests, co-migrating labeled fragments generated in the S. aureus primary digests were cut out from dried gel and submitted to B. subtilis nagarse (Serva) proteolysis directly on a second 15% acrylamide slab gel, according to Claisse et al. (1980).The gels were infiltrated with the scintillator PPO (2,5-diphenyloxazole, Sigma Chemical Co.) as described by Bonner and Laskey (1974), dried, and exposed to Ko-dak X-ray films for one day to several weeks or months.

Changes in the fidelity of mitochondrial translation result in the biosynthesis of a novel polypeptide, V2
Mitochondrial mit -nonsense mutations cob-M2101 and cob-M3041, localized in the COB gene region coding for maturase bi2, arrest the splicing of COB and COX1 genes pre-mRNAs (Kruszewska & Slonimski, 1984b).This is directly reflected in the synthesis of mitochondrial polypeptides since mutant cells do not synthesize Cobp and Cox1p (Jacq et al., 1982).mrf1136 and mrf1-145, previously isolated as nuclear mutations that suppress the respiratory deficiency of cob-M2101, cob-M3041 and several other mit -nonsense mutations localized in different mitochondrial genes, were subsequently identified as alleles of a gene encoding the mitochondrial release factor (Kruszewska & Slonimski, 1984a;Towpik et al., 2004).
To determine the effect of the mrf1 suppressor mutations on the pattern of proteins synthesized in mitochondria of the cob-M2101 mutant, the cells were labeled with Na 2 [ 35 S]O 4 in the presence of cycloheximide to inhibit cytoplasmic protein synthesis.Proteins were then analyzed by SDS/PAGE and autoradiography (Fig. 1).The wild-type CK01 S. cerevisiae cells, grown on media containing glucose or glycerol, synthesize a basic set of mitochondrial polypeptides: Var1p, a mitochondrial ribosomal protein, Cox1p, Cox2p and Cox3p, subunits of cytochrome oxidase, Cobp, the apocytochrome b subunit of coenzyme Q -cytochrome c reductase, and Atp6p, Atp8p and Atp9p, subunits of the F 0 F 1 -ATPase.
In the mit -cells carrying the cob-M2101 mutation both Cob and Cox1 polypeptides are absent but their synthesis is fully restored by mrf1-136 or mrf1-145 suppressor mutations, especially in cells grown on YPGly (Fig. 1B).A novel polypeptide V2, migrating a little slower than Var1p, is present among the mitochondrial translation products in mrf1-136 cells.In contrast to the accumulation of Cox1p, the synthesis of V2p in mrf1-136 mutant cells does not depend on the carbon source (compare Fig. 1A and 1B).This observation points against V2p being a precursor of Cox1p or a mistranslation product of the COX1 gene.
Thus, decreasing the fidelity of stop codon recognition by mutated release factor allows for restoration of the wild type pattern of mitochondrial translation in an ochre mit -but also results in the synthesis of an additional polypeptide.
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, mit -as well as from the mrf1-136 and mrf1-145 cells.The products of mitochondrial translation in organello were analysed by SDS/PAGE and autoradiography (Fig. 2).In contrast to the wild type mitochondria, mitochondria isolated from mrf1-145 cells synthesized V2p after addition of paromomycin.In mrf1-136, V2p was synthesized spontaneously but its synthesis was apparently intensified by paromomycin.This points towards the synergism of mrf1 mutations and the antibiotic in promoting V2p synthesis, making it plausible that V2p represents a product of mistranslation.

V2p is a derivative of Var1 polypeptide
Since mrf1 mutants decrease the fidelity of stop codon recognition (Towpik et al., 2004) we assumed that the V2 polypeptide may have resulted from reading through of the natural termination codon in VAR1 mRNA.This hypothesis was substantiated by direct compari- The wt, mit -and mrf1 strains used were isogenic.
Lanes from left represent: CK01, CK311-cob-M2101, CK247-cob-M3041, CK311/B136 and CK311/B145 (see Table 1 for the strain details).In vivo labeling of mitochondrially synthesized proteins was performed in the presence of Na Radiolabeled polypeptides were synthesized in isolated mitochondria and separated by SDS/PAGE.Var1, V2 and Cox1 proteins were isolated from the gels and submitted to digestion with S. aureus V8 protease.Spontaneously synthesized or paromomycin-induced V2p, both after partial and extensive digestion created patterns almost identical with those obtained for Var1p (Fig. 3A).The peptide patterns obtained with V2p and Var1p, synthesized in the wild-type and suppressor strains, could almost be superimposed.In contrast, the patterns for V2p and Cox1p were entirely different.This further refuted Vol. 52 Translational readthrough in yeast mitochondria 133  The identity of peptides co-migrating in the primary digests of V2p and Var1p was further confirmed by the second digestion.In this respect, peptides No. 2 and 3 from S. aureus V8 protease digests were isolated and treated with Nagarse.In all strains, Var1p-derived peptides were the same as those detected in the V2p-derived products (Fig. 3B).Control digest of a Cox1p-derived peptide migrating in the vicinity of Var1 peptide No. 3 confirmed that the peptide detected in V2p and Var1p is specific for the VAR1 gene product.In conclusion, the peptide map analysis indicates that the V2 product is a derivative of mitochondrial Var1 polypeptide.

DISCUSSION
Yeast mitochondrial ribosomes synthesize eight major polypeptides, seven of which constitute subunits of membrane respiratory complexes (Foury et al., 1998).The eighth product of the mitochondrial genome, a soluble protein Var1, is a mitoribosomal protein (Terpsta et al., 1979).Here we examined the products of mitochondrial translation in the release factor mutants, mrf1-136 and mrf1-145, that affect the fidelity of stop codon recognition (Towpik et al., 2004).This led to observation of the synthesis of a novel protein, identified by finger printing as a Var1p derivative.We suggest that this novel protein is a readthrough product resulting from mistranslation of the regular stop codon in VAR1 mRNA.
The VAR1 gene is unusual in its base composition: it contains nearly 90% AT and a 46 bp GC-rich palindromic cluster (Butow et al., 1985).Furthermore, VAR1 is an expandable gene that participates in a recombinational event resembling exon shuffling, although no intron is present.Strain dependent variations of VAR1 ORF differing in size by up to 7% were observed but this variation does not detectably interfere with its function (Butow et al., 1985).In contrast to other yeast mitochondrial genes, no mutations were isolated in VAR1 except for a deletion of a dodecamer sequence at the 3' end of the mRNA (Butow et al., 1989).Little is known about VAR1 expression.No translational activator specific for VAR1 is known, although the 5' regions of VAR1 mRNA apparently mislocalized the synthesis of Cox2 and Cox3 proteins from chimeric mRNA (Sanchirico et al., 1998).Despite Var1p being the only mitochondrially encoded soluble protein, its synthesis seems to be localized in close proximity to the inner membrane, but at a site distinct from the site of synthesis of Cox2p and Cox3p (Fiori et al., 2003).Membrane associated synthesis of Var1p could help initiate the ribosome assembly process, which would involve all remaining mitoribosomal proteins imported from the cytoplasm (Fiori et al., 2003).A role for Var1p in mitoribosome assembly has been suggested previously (Davis & Ellis, 1995).
In this work we show the possibility of an alternative termination of Var1p biosynthesis.We have analysed mitochondrially synthesized proteins from mrf1-136 and mrf1-145 strains harboring, respectively, the 352 S®I and 216 S®Y amino acid substitutions in the mitochondrial release factor mRF1 (Towpik et al., 2004).A novel polypeptide V2, migrating a little slower than Var1p, is present among the mitochondrial translation products in mrf1-136 cells.An identical longer form of Var1p appears in the mutant mrf1-145, when protein synthesis is probed in the presence of paromomycin.This points towards a synergism of mrf1 mutations and the antibiotic in promoting V2p synthesis, making it plausible that V2p represents a product of VAR1 mistranslation.This hypothesis was substantiated by direct comparison of peptide maps of original Var1p and V2p.
The molecular mass of V2p calculated from SDS-gels exceeded that of Var1p by 1.2-2.3kDa.We favour the idea that V2p is gener-ated as a product of reading through the natural ochre terminator in VAR1 mRNA.Translational suppression of the natural VAR1 ochre terminator with concomitant change in phase could allow reading into short ORFs and stopping at the next ochre terminators detected after 19 (phase +1) or 30 (phase -1) codons.The molecular mass of V2p indicates that its synthesis results from the +1 frameshift.Frameshifting is known to occur at a high rate at specific points during the expression of at least some mitochondrial genes (Fox & Weiss-Brummer, 1980).The change in phase at a natural stop codon, observed here, could be promoted by mrf1 mutations in the release factor gene and by paromomycin.
To understand why V2p is the only readthrough product among mitochondrially synthesized proteins, we examined the sequences preceding and following the natural stop codon in the seven remaining mitochondrial polypeptides.All the stop codons were UAA and the termination regions, except for COX3 mRNA, were AT-rich, but no consensus sequence was found.No specific motif was detected allowing for speculation that the stop codon in VAR1 mRNA is especially susceptible to frameshifting.However, according to theoretical predictions, a +1 frameshift of all the other seven stop codons would result in minute extensions of the synthesized proteins.Such short extensions, even if they existed, would be not discerned by the SDS/PAGE.We showed that the efficiency of V2p synthesis, reflecting the readthrough level, differs for the two mrf1 alleles examined.The higher ambiguity of the mrf1-136 216 S®Y than the mrf1-145 352 S®I mutant is consistent with the spatial localization of the corresponding substituted amino acids.Ser216 is buried in the mRF1 structure in a tight pocket formed by the surrounding residues.Conversion of the serine side chain into the bulkier tyrosine could destroy the internal packing, leading to a local conformational change of the backbone possibly affecting the linker between domains 1 and 2 of mRF1, and, in consequence, the relative orientation of these domains (Towpik et al., 2004).In contrast S352I substitution may only alter the mRF1 interaction with the ribosome.We cannot exclude an alternative termination of Var1p biosynthesis occurring at low frequency in the wild type cell.The readthrough of the natural stop codon was documented for the yeast chromosomal gene PDE2.The synthesis of C-terminally extended Pde2 protein was important for the regulation of the cellular level of cAMP (Namy et al., 2001).It is not known whether the extended form of Var1p has any physiological role.

Figure 1 .
Figure 1.Synthesis of a novel polypeptide, V2, migrating a little slower than Var1p is observed in the mrf1-136 mutant.
cycloheximide.Cells were disrupted by glass beads and the extracts were analyzed on 10-15% gradient SDS/PAGE followed by autoradiography.Cox1, Cox2, Cox3 are subunits 1, 2, 3 of cytochrome c oxidase, respectively; Cob, apocytochrome b; Atp6, Atp8 and Atp9 are subunits of the F 0 F 1 -ATPase; Var1, mitoribosomal protein.A. Cells grown in glucose medium (YPD).B. Cells grown in glycerol medium (YPGly).Lanes 1 and 2 in B represent the results of independent experiments.Respective fragments of gels A and B are shown enlarged.son of peptide maps of original Var1p and V2p.

Table 1 . Saccharomyces cerevisiae strains used in this study
Cells grown as described above were suspended in low sulphate me- -