Yeast Cells Lacking the Mitochondrial Gene Encoding the ATP Synthase Subunit 6 Exhibit a Selective Loss of Complex IV and Unusual Mitochondrial Morphology

Abstract

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8 m . Due to the presence of ARG8 m , accumulation of ؊ / 0 petites issued from large deletions in mtDNA could be restricted to 20 -30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steadystate level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.In the mitochondrial inner membrane, the F 1 F 0 -type ATP synthase produces ATP from ADP and inorganic phosphate by using the energy of the transmembrane electrochemical proton gradient generated by the respiratory chain in the course of electron transfer to oxygen. The ATP synthase harbors two major structural domains, a transmembrane component (F 0 ) containing a proton-permeable pore and a peripheral, matrixlocalized, catalytic component (F 1 ) where the ATP is synthesized (1-4). In the F 0 , the core of the proton channel consists of a ring of c subunits (ten in yeast (4)) and one a subunit (Atp6p). Proton movement through this channel coincides with rotation of the subunit c ring (5-9), which results in conformational changes favoring ATP synthesis in the F 1 (1).Due to its good fermenting capacity the yeast Saccharomyces cerevisiae has been extensively used as a genetic system for the study of the mitochondrial ATP synthase (for reviews see Refs. 10 and 11). As in most eukaryotes, the yeast ATP synthase has a dual genetic origin, nuclear and mitochondrial. The yeast mitochondrial ATP synthase genes (ATP6, ATP9, and ATP8) encode the proton channel subunits a and c (usually referred to in yeast as Atp6p and Atp9p), respectively, and a third F 0 subunit (Atp8p) of unknown function. Dozens of mutations in the nuclear ATP synthase genes have provided much information on their protein products (10, 11). In contrast, only a very few mutants of the mitochondrial ATP synthase genes have been reported. Random generation of respiratory growth-deficient yeast strains issued fro...

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C

“…Yeast Atp6p is typically degraded when it cannot assemble. It is presumed to insert in a late step after assembly of the other ATP synthase subunits [23,45]. When subjected to BN-PAGE analysis the Atp6p-less intermediate easily dissociates into several subcomplexes, among which free F 1 particles [23] (this study, Fig.2A).…”

“…Much more important oxygen consumption deficits (67-85%) were observed in atp6-L252P mitochondria. A respiratory defect, especially at the level of complex IV, is a common property of yeast ATP synthase mutants [23,36]. Not surprisingly, a pronounced decrease in the content of complex IV was observed also in the mutant atp6-L252P whereas the abundance of this complex was almost normal in the atp6-S250P mutant, as revealed by BN-PAGE analysis of mitochondrial protein digitonin-extracts ( Fig.…”

“…One AKY13 and AKY14 clone was crossed to the atp6::ARG8m deletion strain MR10 [23] for the production of clones (called AKY5 and RKY66) harboring the MR10 nucleus and where the ARG8m ORF [24] had been replaced by recombination with the mutated atp6-S250P or atp6-L252P genes. The AKY5 clone was identified by its inability to grow in the absence of an external source of arginine and the ability to grow on respiratory medium.…”

“…The rates of ATP synthesis were determined as described in [23]. For respiration ATP synthesis and transmembrane potential (ΔΨ) measurements, freshly prepared mitochondria were diluted to 0.15 mg/ml in the reaction medium thermostated at 28 °C and containing 10 mM Tris-maleate (pH 6.8), 0.65 M sorbitol, 0.3 mM EGTA, and 3 mM potassium phosphate.…”

“…BN-PAGE, western blotting, pulse labeling of mtDNA encoded proteins were performed as described in [23].…”