title: A genetic screen targeted on the FO component of mitochondrial ATP synthase in Saccharomyces cerevisiae.
abstract: In yeast, the two main F(O) proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the F(O) typically result in loss of mtDNA under the form of ρ(-)/ρ(0) cells. Thus, we have designed a novel genetic strategy to circumvent this problem. It exploits previous findings that a null mutation in the nuclear ATP16 gene encoding ATP synthase subunit δ results in massive and lethal F(O)-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the F(O) can thus, in these conditions, be selected positively as cell viability rescuing events. A first set of seven mutants was analyzed and all showed, as expected, very severe F(O) deficiencies. Two mutants carried nuclear mutations in known genes (AEP1, AEP2) required for subunit c expression. The five other mutations were located in mtDNA. Of these, three affect synthesis or stability of subunit a transcripts and the two last consisted in a single amino acid replacement in subunit c. One of the subunit c mutations is particularly interesting. It consists in an alanine to valine change at position 60 of subunit c adjacent to the essential glutamate of subunit c (at position 59) that interacts with the essential arginine 186 of subunit a. The properties of this mutant suggest that the contact zone between subunit a and the ten subunits c-ring structure only involves critical transient interactions confined to the region where protons are exchanged between the subunit a and the c-ring.
authors: Institut de Biochimie et Génétique Cellulaires, UMR 5095 CNRS, Université Victor Segalen Bordeaux 2, 33077 Bordeaux, France.
authors: François
authors: F
authors: Godard
authors: Emmanuel
authors: E
authors: Tetaud
authors: Stéphane
authors: S
authors: Duvezin-Caubet
authors: Jean-Paul
authors: JP
authors: di Rago
mesh_terms: D020134:Catalytic Domain; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D029701:Saccharomyces cerevisiae Proteins; D006180:Proton-Translocating ATPases