Mitochondrial F1Fo-ATP Synthase Research Articles

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase

Purified soluble bovine mitochondrial F1Fo-ATP synthase contained 2mol of ATP, 2mol of ADP and 6mol of Pi/mol. Incubation of this enzyme with 1mM [32P]Pi caused the exchange of 2mol of Pi/mol of F1Fo-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F1Fo-ATP synthase to a buffer containing 30% (v/v) DMSO and 1mM [32P]Pi resulted in the loss of bound nucleotides with the retention of 1mol of ATP/mol of F1Fo-ATP synthase. Six molecules of [32P]Pi were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [32P]Pi into the bound ATP. Azide did not prevent this [32P]Pi ↔ ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F1Fo-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F1Fo-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphateŐATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound Pi at a second catalytic site. The bound phosphateŐATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

The N‐termini of the α and β subunits at the top of F1 stabilize the energy‐transfer function in the mitochondrial F1Fo ATP synthase

Limited trypsin digestion of isolated F1 removed 15 and 7 amino acids from the N-termini of the alpha and beta subunits respectively and left other subunits untouched as shown by electrophoresis, immunoblotting and protein sequencing. The cooperativity for ATP hydrolysis by soluble F1 was impaired by trypsin digestion. The Km2 obtained from Eadie-Hofstee plots apparently decreased in trypsin-digested F1 but the affinity for adenosine 5'-[beta,gamma-imido]triphosphate (AdoPP[NH]P) and GTP hydrolysis was not influenced. The inhibition of ATP hydrolysis by ADP was attenuated by trypsin digestion. Trypsin digestion of F1 did not affect its capacity to bind to Fo nor did it alter the sensitivity of ATP hydrolysis in the F1Fo reconstituted system to oligomycin and N,N'-dicyclohexylcarbodiimide. The cleavage of the alpha and beta subunits did, on the other hand impair: (a) the ATP-driven proton pumping in the reconstituted F1Fo complex: (b) the inhibition by F1 of passive proton conduction in Fo; (c) the inhibition of passive proton conduction in Fo by AdoPP[NH]P binding to F1. These results show that the limited cleavage of the N-termini of the alpha and beta subunits, located on the top of F1, results in decoupling of catalysis from proton transport. The possible relationship of these observations with the binding change rotatory model of the F1Fo ATP synthase is discussed.

NCA2, a second nuclear gene required for the control of mitochondrial synthesis of subunits 6 and 8 of ATP synthase in Saccharomyces cerevisiae.

Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentable carbon source, due to a dysfunction of the mitochondrial F1-Fo ATP synthase. This defect results from an alteration of the mtDNA-encoded protein synthesis level of subunits 6 and 8 of the Fo sector, due to the simultaneous presence of a mutation in two unlinked nuclear genes. These mutations promote a modification of the expression of the cotranscript ATP8-ATP6 (formerly denoted AAP1-OL12): this mRNA undergoes a maturation at a unique site reaching to two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the relative amount of 5.2 kb cotranscript was greatly lowered. NCA2 was isolated from a wild-type yeast genomic library by genetic complementation. The relative level of the 5.2 kb transcript, as the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1848 nucleotide open reading frame was depicted that encoded an amphiphilic protein of 70,816 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2 kb mRNA but did not abolish the respiratory competence of a wild-type strain. Hybridization analyses indicated that NCA2 is located on chromosome XVI and produces a single 2750 base transcript.

NCA2, a second nuclear gene required for the control of mitochondrial synthesis of subunits 6 and 8 of ATP synthase in Saccharomyces cerevisiae

Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentable carbon source, due to a dysfunction of the mitochondrial F1-Fo ATP synthase. This defect results from an alteration of the mtDNA-encoded protein synthesis level of subunits 6 and 8 of the Fo sector, due to the simultaneous presence of a mutation in two unlinked nuclear genes. These mutations promote a modification of the expression of the cotranscript ATP8-ATP6 (formerly denoted AAP1-OL12): this mRNA undergoes a maturation at a unique site reaching to two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the relative amount of 5.2 kb cotranscript was greatly lowered. NCA2 was isolated from a wild-type yeast genomic library by genetic complementation. The relative level of the 5.2 kb transcript, as the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1848 nucleotide open reading frame was depicted that encoded an amphiphilic protein of 70,816 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2 kb mRNA but did not abolish the respiratory competence of a wild-type strain. Hybridization analyses indicated that NCA2 is located on chromosome XVI and produces a single 2750 base transcript.

NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae.

Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentative carbon source, due to a dysfunctioning of the mitochondrial F1-Fo ATP synthase which results from a relative defect in subunits 6 and 8 of the Fo sector. Both proteins are mtDNA-encoded, but the defect is due to the simultaneous presence of a mutation in two unlinked nuclear genes (NCA2 and NCA3, for Nuclear Control of ATPase) promoting a modification of the expression of the ATP8-ATP6 co-transcript (formerly denoted AAP1-OLI2). This co-transcript matures at a unique site to give two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the 5.2-kb co-transcript was greatly lowered. NCA3 was isolated from a wild-type yeast genomic library by genetic complementation. The level of the 5.2-kb transcript, like the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1011-nucleotide ORF was identified that encodes an hydrophilic protein of 35417 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2-kb mRNA but did not abolish the respiratory competence of a wild-type strain. NCA3 is located on chromosome IV and produces a single 1780-b transcript.