Reversible assembly of the ATP-binding cassette transporter Mdl1 with the F1F0-ATP synthase in mitochondria

The half-ABC transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins. The physiological role of the peptides released from mitochondria is currently not understood. Here, we have analyzed the oligomeric state of Mdl1 in the inner membrane and demonstrate nucleotide-dependent binding to the F(1)F(0)-ATP synthase. Mdl1 forms homo-oligomeric, presumably dimeric complexes in the presence of ATP, but was found in association with the F(1)F(0)-ATP synthase at low ATP levels. Mdl1 binds membrane-embedded parts of the ATP synthase complex after the assembly of the F(1) and F(0) moieties. Although independent of Mdl1 activity, complex formation is impaired upon inhibition of the F(1)F(0)-ATP synthase with oligomycin or N,N'-dicyclohexylcarbodiimide. These results are consistent with an activation of Mdl1 upon dissociation from the ATP synthase and suggest a link of peptide export from mitochondria to the activity of the F(1)F(0)-ATP synthase and the cellular energy metabolism.     

Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase

The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F(1)F(O)-ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial-encoded F(O)-subunit Atp6 after its insertion into the inner membrane. On the other hand and independent of its proteolytic activity, Atp23 promotes the association of mature Atp6 with Atp9 oligomers. This assembly step is thus under the control of two substrate-specific chaperones, Atp10 and Atp23, which act on opposite sides of the inner membrane. Strikingly, both ATP10 and ATP23 were found to genetically interact with prohibitins, which build up large, ring-like assemblies with a proposed scaffolding function in the inner membrane. Our results therefore characterize not only a novel processing peptidase with chaperone activity in the mitochondrial intermembrane space but also link the function of prohibitins to the F(1)F(O)-ATP synthase complex.     

Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ～ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ～ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.     

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.     

The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F(1)-F(0) interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F(1)F(0)-ATP synthase.     

Characterization of ATP12, a yeast nuclear gene required for the assembly of the mitochondrial F1-ATPase.

Mitochondrial F1-ATPase is an oligomeric enzyme composed of five distinct subunit polypeptides. The alpha and beta subunits make up the bulk of protein mass of F1. In Saccharomyces cerevisiae both subunits are synthesized as precursors with amino-terminal targeting signals that are removed upon translocation of the proteins to the matrix compartment. Recently, two different complementation groups (G13, G57), consisting of yeast nuclear mutants with defective F1, have been described. Biochemical analyses indicate that the mutational block in both groups of mutants affects a critical step needed for the assembly of the alpha and beta subunits into the F1 oligomer after their transport into mitochondria. In this study the ATP12 gene representative of the nuclear respiratory-deficient mutant of S. cerevisiae (pet) complementation group G57 has been cloned and the encoded product partially characterized. The ATP12 reading frame is 975 base pairs long and codes for a protein of Mr = 36,587. The ATP12 protein is not homologous to the subunits of F1 whose sequences are known, nor does it exhibit significant primary structure similarity to any known protein. In vitro import assays indicate that ATP12 protein is synthesized as a precursor approximately 3 kDa larger than the mature protein. The mitochondrial localization of the protein has been confirmed by Western blot analysis of mitochondrial proteins with an antibody against a hybrid protein expressed from a trpE-ATP12 fusion. Fractionation of mitochondria indicates further that the ATP12 protein is either a minor component of the matrix compartment or is weakly bound to the matrix side of the inner membrane. The molecular weight of the native protein, estimated from its sedimentation properties in sucrose gradients, is at least two times larger than the monomer. This suggests that the ATP12 protein is probably part of a larger complex.     

Mitochondrial consumption of cytosolic ATP: not so fast

In various pathologic circumstances depolarized mitochondria are thought to precipitate cell death by avidly consuming cytosolic ATP. However, for as long as the inner mitochondrial membrane remains intact the reversal potentials of the adenine nucleotide translocase (ANT) and that of F(0)-F(1) ATP synthase are strategically positioned so that they oppose import of cytosolic ATP into the matrix of respiration-impaired mitochondria. This arrangement also seems to protect against a hysteretic consumption of cytosolic ATP accumulating in the mitochondrial matrix, in view of the depolarization caused by inhibition of F(0)-F(1) ATP synthase by the endogenous protein IF1, yielding fast ANT reversal rates.     

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.     

Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase

Eukaryotic cells require mitochondrial compartments for viability. However, the budding yeast Saccharomyces cerevisiae is able to survive when mitochondrial DNA suffers substantial deletions or is completely absent, so long as a sufficient mitochondrial inner membrane potential is generated. In the absence of functional mitochondrial DNA, and consequently a functional electron transport chain and F(1)F(o)-ATPase, the essential electrical potential is maintained by the electrogenic exchange of ATP(4-) for ADP(3-) through the adenine nucleotide translocator. An essential aspect of this electrogenic process is the conversion of ATP(4-) to ADP(3-) in the mitochondrial matrix, and the nuclear-encoded subunits of F(1)-ATPase are hypothesized to be required for this process in vivo. Deletion of ATP3, the structural gene for the gamma subunit of the F(1)-ATPase, causes yeast to quantitatively lose mitochondrial DNA and grow extremely slowly, presumably by interfering with the generation of an energized inner membrane. A spontaneous suppressor of this slow-growth phenotype was found to convert a conserved glycine to serine in the beta subunit of F(1)-ATPase (atp2-227). This mutation allowed substantial ATP hydrolysis by the F(1)-ATPase even in the absence of the gamma subunit, enabling yeast to generate a twofold greater inner membrane potential in response to ATP compared to mitochondria isolated from yeast lacking the gamma subunit and containing wild-type beta subunits. Analysis of the suppressing mutation by blue native polyacrylamide gel electrophoresis also revealed that the alpha(3)beta(3) heterohexamer can form in the absence of the gamma subunit.     

Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues.

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.     

Assembly factors of F1FO-ATP synthase across genomes

Work with respiration-deficient strains of Saccharomyces cerevisiae has provided evidence that assembly of the mitochondrial ATP synthase is dependent on proteins that serve substrate-specific, chaperone-type functions: Atp10p, Atp11p, Atp12p, Atp22p, and Fmc1p. Atp11p and Atp12p mediate the formation of the F1 moiety via interaction with subunits F1-beta and F1-alpha, respectively. The role of Fmc1p is less clear. Atp10p and Atp22p are essential for the formation of the F(O) part, during which Atp10p assists in the incorporation of the F(O)-a subunit. Here we present a comprehensive analysis of ATP synthase assembly factors from all available genomes. The mechanism of the F1 assembly is preserved in all eukaryotic lineages that are capable of ATP synthesis via oxidative phosphorylation and requires Atp11p and Atp12p. Conversely, composition of the F(O) part as well as its assembly is more versatile. We found two distinct subtypes of the F(O)-a subunit, one of which seems to be dependent on the action of Atp10p while the other does not. Restricted occurrence of Fmc1p and Atp22p suggests the existence of lineage-specific assembly factors. Our phylogenetic data served as a source for comparative sequence analysis, which identified evolutionarily conserved residues, putative functional domains and their basic structural features for Atp10p, Atp11p, and Atp12p orthologs. These results provide the basis for detailed molecular analysis of the ATP synthase-specific chaperones.     

Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes

The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria.     

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha‐subunit of F1‐ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria‐rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria‐less, endoplasmic reticulum (ER)‐rich cortical region and the mitochondria‐rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria‐enriched fraction, a considerable amount of ATPα and F1‐ATP synthase beta‐subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1‐ATP synthase gamma‐subunit (ATPγ) and F0‐ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.     

Functional analysis of subunit e of the F1Fo-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region

Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.     

Mitochondrial ATP synthase complex. Membrane topography and stoichiometry of the F0 subunits.

The topography of the subunits of the membrane sector F0 of the ATP synthase complex in the bovine mitochondrial inner membrane was studied with the help of subunit-specific antibodies raised to the F0 subunits b, d, 6, F6, A6L, OSCP (oligomycin-sensitivity-conferring protein), and N,N' -dicyclohexylcarbodiimide (DCCD)-binding proteolipid and to the ATPase inhibitor protein (IF1) as an internal control. Exposure of F0 subunits in inverted and right-side-out inner membranes was investigated by direct antibody binding as well as by susceptibility of these subunits to degradation by various proteases as monitored by gel electrophoresis of the membrane digests and immunoblotting with the subunit-specific antibodies. Results show that subunits b, d, F6, A6L (including its C-terminal end) and OSCP were exposed on the matrix side. Sufficient masses of these subunits to recognize antibodies or undergo proteolysis were not exposed on the cytosolic side. This was also the case for subunit 6 and the DCCD-binding proteolipid on either side of the inner membrane. Quantitative immunoblotting in which bound radio-activity from [125I]protein A was employed to estimate the concentration of an antigen in a sample allowed the determination of the stoichiometry of several F0 subunits and IF1 relative to F1-ATPase. Results showed that per mol of F1 there are in bovine heart mitochondria 1 mol each of d, OSCP, and IF1, and 2 mol each of b and F6. Subunit 6 and the DCCD-binding proteolipid could not be quantitated, because the former transferred poorly to nitrocellulose and the latter's antibody did not bind [125I]protein A.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Increased expression of the F(1)F(o) ATP synthase in response to iron in heart mitochondria

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the alpha, beta, and d subunits of F(1)F(o) ATP synthase increased along with the loss of ATP. This suggests that the F(1)F(o) ATP synthase participates in iron metabolism.