A “Petite Obligate” Mutant of Saccharomyces cerevisiae: FUNCTIONAL mtDNA IS LETHAL IN CELLS LACKING THE δ SUBUNIT OF MITOCHONDRIAL F1-ATPASE

Stéphane Duvezin-Caubet,Malgorzata Rak,Linnka Lefebvre-Legendre,Emmanuel Tetaud,Nathalie Bonnefoy,Jean-Paul Di Rago

doi:10.1074/jbc.m513805200

Abstract

Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.

Highlights

The F1 domain comprises five different subunits, all nucleus-encoded, with ␣3␤3␥1␦1␧1 stoichiometry
Fox and co-workers [20] have developed elegant approaches for the study of mitochondria based on the insertion into the mitochondrial DNA (mtDNA) of nonrespiratory genes like ARG8m. This gene is a mitochondrial version of the nuclear ARG8 gene encoding a mitochondrial protein involved in arginine biosynthesis
Arginine was required for SDC12 to grow in the presence of ethidium bromide, an intercalating agent inducing the loss of mtDNA (Fig. 1C )

Summary

Introduction

The F1 domain comprises five different subunits, all nucleus-encoded, with ␣3␤3␥1␦1␧1 stoichiometry. In apparent contradiction with this idea, a lack in the third central stalk subunit (␧) results in a major F0-mediated mitochondrial uncoupling, only partial effects were observed on the mtDNA in ⌬␧ mutant [18]. Cultures of this mutant usually contain 30% ␳ϩ cells, showing that retention of functional mtDNA in ⌬␧ yeast is not lethal.

Methods

Results

Conclusion