A genetic screen targeted on the FO component of mitochondrial ATP synthase in Saccharomyces cerevisiae

Abstract

In yeast, the two main FO proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the FO typically result in loss of mtDNA under the form of ρ-/ρ0 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 FO-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the FO 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 FO 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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.