F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

Abstract

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F(1) ATPase (or assembly intermediates thereof). Mutants lacking the alpha or beta subunits of F(1), or the Atp11p and Atp12p chaperones that promote F(1) assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. F(1) mutants are also unable to express ARG8(m) when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by F(1) is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of F(1). These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by F(1) to achieve a balanced output of two compartmentally separated sets of ATP synthase genes.

Fig. 1.

In vivo labeling of mitochondrial gene products in wild-type and F₁ mutants. The respiratory competent strains W303 and MR6, and F₁ mutants with the indicated genotypes were grown in rich galactose medium and labeled for 20 min with [³⁵S]methionine + [³⁵S]cysteine in the presence of cycloheximide. Total cellular extracts were separated by SDS/PAGE in two different polyacrylamide gels prepared with a 30:0.8 ratio of acrylamide and bisacrylamide. Upper gel: 12% polyacrylamide gel containing 4 M urea and 25 glycerol. Lower gel: 17.5% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and exposed to X-ray film. The bands corresponding to Atp6p and Atp8p were quantified with a phoshorimager and normalized to Cox3p. The averages of 4–5 independent experiments are expressed as percentage of wild-type.

Fig. 2.

ATP8/6 mRNAs are normally transcribed and processed in F₁ mutants. (A) Diagram showing transcription of the primary polycistronic COX1/ATP8/ATP6 RNA from a site upstream of COX1. Cleavage sites that produce the mature messengers are shown by asterisks. The 5.2- and 4.6-kb mRNAs are the result of cleavages at L and S, respectively. COX1, which contains multiple introns in the W303 strain, is not drawn to scale. (B) Mitochondrial RNAs isolated from W303 and Δatp11, Δatp12, and Δatp2 mutants were separated on a 1% agarose gel, stained with ethidium bromide (left panel) and transferred to a Nytran membrane that was hybridized with ³²P labeled ATP6- and COX3-specific probes. The ATP6/ATP8 mRNAs was quantified with a phosphorimager and normalized to the COX3 mRNA.

Fig. 3.

F₁ mutants do not express ARG8^m from the ATP6 or ATP8 locus of mitochondrial DNA. The parental strain MR6 (Δarg8 [ATP6 ATP8]) and the different respiratory deficient mutants with either the mitochondrial Δatp6::ARG8^m or Δatp8::ARG8^m allele were grown overnight in minimal glucose medium supplemented with auxotrophic requirements including arginine. Serial dilutions were spotted on minimal glucose with or without arginine and incubated at 30 °C for 2 days. The percentage of ρ⁻ or ρ⁰ mutants in the cultures is indicated next to each strain in the right hand margin. The mitochondrial genotypes are enclosed by the straight brackets.

Fig. 4.

ATP6 and ATP8 are expressed in a catalytic inactive F₁ mutant. (A) The parental strain D273–10B/A1 and the point mutants P159 (atp2-A192V) were labeled in vivo as in Fig. 1 and separated by SDS/PAGE on a 12% gel containing 6 M urea and 25% glycerol (left panel) and a 17.5% polyacrylamide gel (right panel). (B) The point mutant (atp2-A192V Δarg8) with wild-type mtDNA and with the Δatp6::ARG8^m allele were grown and spotted on minimal glucose with or without arginine as in Fig. 3.

Fig. 5.

Characterization of F₁ and expression of Atp6p and Atp8p in γ, ε, and δ subunit null mutants. (A) Mitochondria were extracted with 2% digitonin and samples representing 250 μg of starting mitochondrial protein were analyzed by BN-PAGE as described in Materials and Methods. (B) The respiratory competent strain W303 and the ε null mutant (Δatp15) were labeled in vivo and separated on two different SDS/PAGE gel systems as in Fig. 1.

Fig. 6.

Expression of Atp6p, Atp8p in F₀ and peripheral stalk mutants. (A) Mitochondrial gene products of the parental strain MR6 and of the Δatp8::ARG8^m mutant were labeled in vivo and analyzed by SDS/PAGE as in Fig. 1. (B) The respiratory competent parental strains MR6 and D273–10B/A1, the subunit b, d, and h null mutants (Δatp4, Δatp7, and Δatp14, respectively) and the subunit b and h point mutants (atp4 and atp14) (20) were labeled and analyzed as in Fig. 1. (C) Strains with the indicated genotypes were serially diluted, spotted on minimal glucose with or without arginine and grown as in Fig. 3.

Fig. 7.

Suppression of the Atp6p and Atp8p translation defect in F₁ mutants by ATP22. (A) Mutants with the indicated genotypes were transformed with ATP12 and ATP22 cloned in high-copy plasmids and were grown overnight in minimal glucose supplemented with all of the amino acids requirements including arginine. Serial dilutions were spotted on minimal glucose with or without arginine and grown as in Fig. 3. (B) ARG8^m is expressed from the ATP8 but not ATP6 locus. The parental strain MR6 (Δarg8 [ATP6 ATP8]) and the different mutants were grown in minimal glucose containing arginine and serial dilutions were spotted on minimal medium with and without arginine as in Fig. 3. (C) ATP22 restores translation of Atp6p and Atp8p in an F₁ mutant. The respiratory competent strain W303, the Δatp12 mutant and transformants harboring ATP12 and ATP22 on high copy plasmids were labeled in vivo as in Fig. 1 and separated by SDS/PAGE on a 12% gel containing 6 M urea (upper panel) and a 17.5% polyacrylamide gel (lower panel).