Development of Dual Control Allotopic Expression System for Subunit 8 of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase

Abstract Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the α- and γ-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

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Expression of Bovine F1-ATPase with Functional Complementation in Yeast Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.m411113200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22418-22424 ◽

Cited By ~ 8

Author(s):

Neeti Puri ◽

Jie Lai-Zhang ◽

Scott Meier ◽

David M. Mueller

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mass ◽

Atp Synthase ◽

Specific Activity ◽

Functional Complementation ◽

Yeast Saccharomyces Cerevisiae ◽

F1 Atpase ◽

Mitochondrial Atp Synthase ◽

Genes Encoding ◽

Molecular Machinery

The mitochondrial F1F0-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of ∼600 kDa. F1-ATPase is composed of α3β3γδϵ with an overall molecular mass of 370 kDa. The genes encoding bovine F1-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (ΔαΔβΔγΔδΔϵ). This strain expressing bovine F1 is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F1-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F1- or F1F0-ATP synthase.

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Isolation and complete amino acid sequence of the mitochondrial ATP synthase epsilon-subunit of the yeast Saccharomyces cerevisiae.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)35231-6 ◽

1991 ◽

Vol 266 (2) ◽

pp. 723-727

Author(s):

G Arselin ◽

J C Gandar ◽

B Guérin ◽

J Velours

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Sequence ◽

Atp Synthase ◽

Yeast Saccharomyces Cerevisiae ◽

Complete Amino Acid Sequence ◽

Epsilon Subunit ◽

Mitochondrial Atp Synthase

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A model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

10.1101/2021.07.13.452228 ◽

2021 ◽

Author(s):

Jean-Pierre Mazat ◽

Anne Devin ◽

Edgar Yoboue ◽

Stephane Ransac

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Atp Synthase ◽

Yeast Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Complex Iv ◽

Mitochondrial Atp Synthase ◽

Atp6 Gene ◽

Substrate Level

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-C-oxidase preventing yeast growth on oxidative substrates. The overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate in stimulating the substrate-level phosphorylation of ADP cou-pled to conversion of α-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.

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