A “Petite Obligate” Mutant of Saccharomyces cerevisiae

Abstract

Within the mitochondrial F 1 F 0 -ATP synthase, the nucleus-encoded ␦-F 1 subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the ␦ subunit gene (⌬␦) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% ؊ /°petites (i.e. cells missing a large portion (>50%) of the mtDNA ( ؊ ) or totally devoid of mtDNA (°)). Previous work has suggested that the absence of complete mtDNA ( ؉ ) in ⌬␦ 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 ⌬␦ yeast. We further show that combined with a nuclear mutation (⌬atp4) preventing the ATP synthase proton channel assembly, a lack of ␦ subunit fails to destabilize the mtDNA, and ؉ ⌬␦ cells become viable. We conclude that ⌬␦ yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the ؊ /°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.The mitochondrial inner membrane contains the ATP synthase, which utilizes a transmembrane proton gradient to catalyze ATP synthesis from inorganic phosphate and ADP. The ATP synthase has two major structural domains, an F 0 component, which forms a protonpermeable pore across the membrane, and a peripheral, matrix-localized, F 1 component, where the ATP is synthesized (1).The F 1 domain comprises five different subunits, all nucleus-encoded, with ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 stoichiometry. The three ␣ subunits and the three ␤ subunits alternate in position within a hexamer that contains the adenine nucleotide processing sites (2-4). The ␥, ␦, and subunits form a subcomplex of F 1 , named the central stalk, linking the ␣ 3 ␤ 3 subcomplex to the ATP synthase proton channel (5). During catalysis, the central stalk rotates together with a transmembrane ring of 10 -12 c subunits in the F 0 (6 -12). In the course of this rotation, the ␥ subunit sequentially interacts with the three ␣␤ pairs in a way that favors ATP synthesis in the catalytic sites, as required by the binding change mechanism (1).In the yeast Saccharomyces cerevisiae, null mutations of the ␦ or ␥ subunit massively destabilize the mitochondrial genome in the form of 100% cytoplasmic petites, which are cells with a large (Ͼ50%) deletion in mitochondrial DNA (mtDNA) 6 ( Ϫ ) or completely lacking mtDNA (°) (13,14). This is an intriguing observation, since many other mutations impairing mitochondrial oxidative phosphorylation, including a null ...

A Genetic Screen Targeted on the FO Component of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

“…A lack of Atp4p prevents the incorporation of Atp6p into the F O (37). In these conditions, the absence of subunit ␦ can no longer uncouple the mitochondrial membrane and subunit ␦-deficient cells with a complete mtDNA are viable (25). Transformation of ⌬␦ ϩ ⌬atp4 cells with a plasmid-borne wild-type ATP4 gene will restore the assembly of a functional F O and the reconstituted ⌬␦ cells will die.…”

“…F O -Retention of functional mtDNA in ⌬␦ cells is not viable due to massive F O -mediated proton leaks across the inner mitochondrial membrane (25). As a result, 100% of ⌬␦ cells are found as Ϫ / 0 petites (22).…”

“…However, if the ⌬␦ ϩ ⌬atp4 cells acquired a mutation that alone inactivates the F O prior to their transformation with ATP4, the reconstituted ⌬␦ cells are expected to be viable. To eliminate the Ϫ / 0 mutation from the screen, we used cells with modified mtDNA in which ARG8 m , a non-respiratory genetic marker (27), has been integrated into an intergenic region (25). This is a mitochondrial version of the nuclear gene ARG8 encoding a mitochondrial protein involved in arginine biosynthesis (27).…”

“…Using a regulatable subunit ␦ gene (ATP16), we have shown that a lack in this subunit results in the formation of partial F 1 F O assemblies freely transporting protons across the mitochondrial inner membrane (24). Beyond a 50% deficit in subunit ␦, the cells are unable to maintain any electrical potential (⌬⌿) across the mitochondrial inner membrane (25). As a consequence, the cells need then to inactivate the F O to survive, which explains the 100% conversion into Ϫ / 0 cells of strains lacking the subunit ␦ gene.…”

“…The screen is thus positive and targeted on the F O . To avoid rescue of ␦-deficient cells by the Ϫ / 0 mutation, which is much more frequent than specific mutations of the F O (ϳ10 Ϫ2 versus Ͻ10 Ϫ5 ), we used a host strain in which a non-respiratory genetic marker, ARG8 m (27), has been integrated into an intergenic region of the mitochondrial genome (25). This is a mitochondrial version of the nuclear gene ARG8 that encodes a mitochondrial protein involved in arginine biosynthesis (27 …”