Aep3p Stabilizes the Mitochondrial Bicistronic mRNA Encoding Subunits 6 and 8 of the H+-translocating ATP Synthase of Saccharomyces cerevisiae

Timothy P Ellis,Kevin G Helfenbein,Alexander Tzagoloff,Carol L Dieckmann

doi:10.1074/jbc.m314162200

Timothy P Ellis, Kevin G Helfenbein + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m314162200

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Abstract

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

Highlights

The steady-state levels of ATP8/6 RNAs are affected by the products of three nuclear genes
AEP3 was identified by a twostep analysis of unknown yeast proteins in the Saccharomyces Genome Database (SGD) designed to identify mitochondrial proteins involved in RNA metabolism
A sequential screen of all yeast ORFs for those encoding mitochondrial proteins, followed by a search for sequence motifs commonly found in RNA-interacting proteins, led to the identification of AEP3 as a strong candidate for a mitochondrial protein involved in RNA metabolism

Summary

Synthase of Saccharomyces cerevisiae*

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to ϳ35% that of wild type. In Saccharomyces cerevisiae, subunits 6, 8, and 9 of the F0 domain are encoded by the mitochondrial genome. Processing of the initial transcript yields two major mRNAs containing both ATP8 and ATP6 (and ENS2, if present in the genome) [4]. Of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721. We have used an HAtagged version of Aep3p to localize the protein to the matrix side of the inner mitochondrial membrane

EXPERIMENTAL PROCEDURES

RESULTS

Mitochondrial rhoϩ rhoo rhoϩ rhoia rhoia rhoia rhoo

DISCUSSION

Findings

ATPase ϩoligomycin