Assembled ATP Synthase Research Articles

Cooperativity of gating of ATP synthase and ant ion channels.

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. Our studies of highly purified, fully assembled ATP synthase monomers demonstrate that ATP synthase forms large conductance, Ca2+-sensitive and voltage-gated channels consistent with the known biophysical properties of mPTP. We have previously characterized a non-selective uncoupling channel within the ATP synthase c-subunit ring, the ATP synthase c-subunit leak channel (ACLC), the persistent opening of which initiates cell death. We have growing evidence for the role of ACLC in mitochondrial permeability transition (mPT), including reversible and irreversible gating of ACLC by the F1 as its inactivation gate. We observe dissociation of ATP synthase F1 from FO during neuronal glutamate toxicity, suggesting that non-reversible dissociation of F1 from FO occurs in pathology. However, studies of other laboratories suggest that the adenine nucleotide transporter (AAC or ANT) also forms a leak and death channel with similar biophysical characteristics to that of mPTP but different pharmacological characteristics from ACLC. Therefore, we used pharmacology to determine if ANT and ACLC cooperate in formation of the mPT pore. We isolated submitochondrial vesicles (SMVs; ATP synthasomes) from rat liver and reconstituted the SMVs with giant lipid vesicles to form larger proteoliposomes for patch clamping. “Live” antibody imaging reveals that both ANT and ATP synthase are present in the lipid vesicles. Patch clamp recordings demonstrate a multi-conductance, voltage gated channel with the biophysical and pharmacological characteristics of mPTP. Pharmacological inhibition of ANT and ATP synthase respectively demonstrates cooperative gating suggesting reciprocal interaction and/or cross-inhibition between the two channels. These findings suggest that the mPTP is comprised of regulated pores located within the ACLC and ANT, gating in a cooperative manner.

Biochemical thresholds for pathological presentation of ATP synthase deficiencies

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15–80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10–30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

Persistence of the permeability transition pore in human mitochondria devoid of an assembled ATP synthase

The opening of the permeability transition pore, a nonspecific channel in inner mitochondrial membranes, is triggered by an elevated total concentration of calcium ions in the mitochondrial matrix, leading to disruption of the inner membrane and necrotic cell death. Cyclosporin A inhibits pore opening by binding to cyclophilin D, which interacts with the pore. It has been proposed that the pore is associated with the ATP synthase complex. Previously, we confirmed an earlier observation that the pore survives in cells lacking membrane subunits ATP6 and ATP8 of ATP synthase, and in other cells lacking the enzyme's c8 rotor ring or, separately, its peripheral stalk subunits b and oligomycin sensitive conferral protein. Here, we investigated whether the pore is associated with the remaining membrane subunits of the enzyme. Individual deletion of subunits e, f, g, and 6.8-kDa proteolipid disrupts dimerization of the complex, and deletion of DAPIT (diabetes-associated protein in insulin sensitive tissue) possibly influences oligomerization of dimers, but removal of each subunit had no effect on the pore. Also, we removed together the enzyme's membrane bound c8 ring and the δ-subunit from the catalytic domain. The resulting cells assemble only a subcomplex derived from the peripheral stalk and membrane-associated proteins. Despite diminished levels of respiratory complexes, these cells generate a membrane potential to support uptake of calcium into the mitochondria, leading to pore opening, and retention of its characteristic properties. It is most unlikely that the ATP synthase, dimer or monomer, or any component, provides the permeability transition pore.

Mitochondrial Megachannel Resides in Monomeric ATP Synthase

Mitochondrial ATP synthase has been shown recently to be vital not only for cellular energy production but also for energy dissipation and cell death. We identified and characterized a large non-selective uncoupling channel within the ATP synthase c-subunit ring, the persistent opening of which initiates cell death. We have continuing evidence for the crucial role of this channel in mitochondrial permeability transition (mPT). We have now purified ATP synthase from porcine heart mitochondria and performed single-channel studies. Excised proteoliposome patch-clamp recordings demonstrate that highly pure and fully assembled ATP synthase monomers form large conductance, Ca2+-sensitive and voltage-gated channels. We confirmed the monomeric state of ATP synthase by cryo-electron microscopy studies of ATP synthase reconstituted into proteoliposomes. We have also heterologously overexpressed and purified human ATP synthase c-subunit from E. coli plasma membranes. We show that human c-subunit purified from bacteria forms large conductance channels identical to those purified from HEK-293 cells. The channel is gated by polar amino acid residues situated at the mouth of the pore and by the hydrophilic F1 portion of ATP synthase. We find that dissociation of ATP synthase F1 from FO occurs when we expose primary hippocampal neurons to glutamate toxicity, suggesting that the non-reversible dissociation of F1 from FO is pathological. We have successfully knocked out five of the six alleles of the three genes encoding ATP synthase c-subunit in mouse embryonic stem cells by CRISPR-Cas9, which resulted in only 10% of the total c-subunit expression. Patch-clamp recordings of mitoplasts isolated from these cells demonstrate low conductance activity that is poorly calcium responsive. These findings confirm that the largest of all inner mitochondrial membrane conductances resides within the ATP synthase monomer, more specifically within its membrane-embedded c-subunit ring.

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.

Knockout of Tmem70 alters biogenesis of ATP synthase and leads to embryonal lethality in mice.

TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at ∼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.

Interaction between PVY HC-Pro and the NtCF1β-subunit reduces the amount of chloroplast ATP synthase in virus-infected tobacco.

The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600 μmol m−2 s−1. Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1β-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn’t interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1β-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions.

Mitochondrial membrane assembly of TMEM70 protein

Dysfunction of TMEM70 disrupts the biogenesis of ATP synthase and represents the frequent cause of autosomal recessive encephalocardiomyopathy. We used tagged forms of TMEM70 and demonstrated that it has a hairpin structure with the N- and C-termini oriented towards the mitochondrial matrix. On BN-PAGE TMEM70 was detected in multiple forms including dimers and displayed partial overlap with assembled ATP synthase. Immunoprecipitation studies confirmed mutual interactions between TMEM70 molecules but, together with immunogold electron microscopy, not direct interaction with ATP synthase subunits. This indicates that the biological function of TMEM70 in the ATP synthase biogenesis may be mediated through interaction with other protein(s).

Three Distinct Isoforms of ATP Synthase Subunit c Are Expressed in T. brucei and Assembled into the Mitochondrial ATP Synthase Complex

One striking feature of the biology of trypanosomes is the changes in mitochondrial structure and function that occur as these parasites transition from one life cycle stage to another. Our laboratory has been interested in the role the mitochondrial ATP synthase plays in mitochondrial changes through the life cycle. Analysis of the recently completed T. brucei genome suggested that there may be multiple putative genes encoding ATP synthase subunit c. While homologous in their 3′ ends, these genes differ in their 5′ ends and, if expressed, would result in three distinct proteins. Our analysis showed that all three of the possible transcripts were detected in both procyclic and bloodstream stages, although the c-3 transcript was less abundant than that for c-1 or c-2. The three isoforms of subunit c are produced in both the bloodstream and procyclic stages and their mature protein products possess distinct N-terminal regions of the protein as found within mitochondria. All three isoforms are also incorporated into the assembled ATP synthase complex from procyclic cells. Although multiple subunit c genes have been found in other organisms, they produce identical polypeptides and the finding of significant differences in the mature proteins is unique to T. brucei.

TMEM70: a mutational hot spot in nuclear ATP synthase deficiency with a pivotal role in complex V biogenesis

Mammalian complex V (F1F0-ATP synthase or ATPase) uses the proton gradient to generate ATP during oxidative phosphorylation and requires several helper proteins, including TMEM70, to form the holoenzyme in a stepwise process in which nuclear DNA is combined with mitochondrial DNA-encoded subunits. We report the clinical and molecular findings in three patients presenting lactic acidosis, 3-methylglutaconic aciduria, and hypertrophic cardiomyopathy. All three showed an isolated defect of fully assembled ATP synthase in association with a "common" (c.317-2A > G) and a new (c.628A > C/p.T210P) variant in TMEM70. Interestingly, one of the patients also showed nitric oxide-responsive pulmonary arterial hypertension, a finding never before associated with TMEM70 deficiency. In addition to widening the clinical and mutational spectrum of defective ATP synthase, our study also suggests that mutant TMEM70 associates in high molecular weight complexes (470-550kDa) when expressed in Hela cells and exerts a direct action in ATP synthase biogenesis and assembly, mediating the incorporation of F1 moieties.

Modular assembly of yeast mitochondrial ATP synthase

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.

Expression and processing of the TMEM70 protein

TMEM70 protein represents a novel ancillary factor of mammalian ATP synthase. We have investigated import and processing of this factor in human cells using GFP- and FLAG-tagged forms of TMEM70 and specific antibodies. TMEM70 is synthesized as a 29 kDa precursor protein that is processed to a 21 kDa mature form. Immunocytochemical detection of TMEM70 showed mitochondrial colocalization with MitoTracker Red and ATP synthase. Western blot of subcellular fractions revealed the highest signal of TMEM70 in isolated mitochondria and mitochondrial location was confirmed by mass spectrometry analysis. Based on analysis of submitochondrial fractions, TMEM70 appears to be located in the inner mitochondrial membrane, in accordance with predicated transmembrane regions in the central part of the TMEM70 sequence. Two-dimensional electrophoretic analysis did not show direct interaction of TMEM70 with assembled ATP synthase but indicated the presence of dimeric form of TMEM70. No TMEM70 protein could be found in cells and isolated mitochondria from patients with ATP synthase deficiency due to TMEM70 c.317-2A>G mutation thus confirming that TMEM70 biosynthesis is prevented in these patients.

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.

The Cardiolipin Transacylase, Tafazzin, Associates with Two Distinct Respiratory Components Providing Insight into Barth Syndrome

Mutations in the mitochondrial cardiolipin (CL) transacylase, tafazzin (Taz1p), result in the X-linked cardioskeletal myopathy, Barth syndrome (BTHS). The mitochondria of BTHS patients exhibit variable respiratory defects and abnormal cristae ultrastructure. The biochemical basis for these observations is unknown. In the absence of its target phospholipid, CL, a very large Taz1p complex is missing, whereas several discrete smaller complexes are still observed. None of the identified Taz1p complexes represents Taz1p homodimers. Instead, yeast Taz1p physically assembles in several protein complexes of distinct size and composition. The ATP synthase and AAC2, both required for oxidative phosphorylation, are identified in separate stable Taz1p complexes. In the absence of CL, each interaction is still detected albeit in reduced abundance compared with when CL is present. Taz1p is not necessary for the normal expression of AAC2 or ATP synthase subunits or assembly of their respective complexes. In contrast, the largest Taz1p complex requires assembled ATP synthase and CL. Mitochondria in Delta taz1 yeast, similar to ATP synthase oligomer mutants, exhibit altered cristae morphology even though ATP synthase oligomer formation is unaffected. Thus, the Taz1p interactome defined here provides novel insight into the variable respiratory defects and morphological abnormalities observed in mitochondria of BTHS patients.

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F1 and F0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.

Functional characterization of Chlamydomonas reinhardtii with alterations in the atpE gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the epsilon subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the epsilon subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the epsilon subunit, and much lower levels of the alpha and beta subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the epsilon subunit, which is expressed normally in the cell.

Lysine 43 Is Trimethylated in Subunit c from Bovine Mitochondrial ATP Synthase and in Storage Bodies Associated with Batten Disease

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.

A novel fluorescent marker for assembled mitochondria ATP synthase of yeast: OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo

We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria. The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus. Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol. Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6. Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies. The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP. In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET).

Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases

To study the assembly of mitochondrial F 1F 0 ATP synthase, cultured human cells were labeled with [ 35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F 1 part appeared to be assembled, and next an intermediate product that contained F 1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochondrial diseases.

The chloroplast ATP synthase in Chlamydomonas reinhardtii: II. Biochemical studies on its biogenesis using mutants defective in photophosphorylation

We have carried out an analysis of the synthesis, cellular accumulation, and membrane binding of the chloroplast-encoded subunits of the ATP synthase (α, β, ϵ, I, III, and IV) in several mutants of Chlamydomonas reinhardtii defective in photophosphorylation. These data gave some insight on the putative genetic lesion in each mutant and allowed some characterization of the assembly and stabilization of the ATP synthase complex in the thylakoid membranes. Four chloroplast mutants are likely to be altered in chloroplast structural genes coding for coupling factor (CF) 1 subunits β and ϵ and for CF0 subunits I and IV. A fifth chloroplast mutant and three nuclear mutants were altered in genes regulating either transcription or translation of chloroplast genes coding for CF1 subunits α and β and CF0 subunits III and IV. Evidence is presented (i) for a control of the rate of synthesis of subunit β by subunit α in the absence of ATP synthase assembly and (ii) for an interaction between α and β subunits in the stroma of the chloroplast which protects α subunits from proteolytic degradation. The role of several chloroplast-encoded subunits of CF0 and CF1 in the stabilization of partially assembled ATP synthase is discussed. We conclude that in the absence of ATP synthase assembly, CF0 cannot accumulate in the thylakoid membranes, whereas α and β subunits, presumably engaged in soluble CF1, can accumulate in the stroma of the chloroplast.