Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane.

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein.     

Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation.

The skeletal muscle mitochondrial uncoupling protein-3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (DYm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was contrasts with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered DYm, ATP, and the ATP/ADP ratio. UCP3-overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3-overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2-Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering DYm and enhanced fatty acid-dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up-regulated in metabolic stress situations where it could be involved in nutrient partitioning.     

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.     

Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane

Submitochondrial particles were labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide. On sodium dodecyl sulfate-polyacrylamide gels, eosin fluorescence occurred in a single band of Mr approximately 30,000. The labeled band was identified as the ADP/ATP translocator, since EMA binding was completely inhibited by carboxyatractylate. Furthermore, the EMA-labeled polypeptide had the same molecular weight as the purified carboxyatractylate-bound translocator and the purified EMA-labeled translocator. Rotational diffusion of the translocator around the membrane normal in submitochondrial particles was measured by observing flash-induced absorption anisotropy of EMA. The translocator rotates with a time constant which varied from approximately 240 microseconds at 5 degrees C to approximately 100 microseconds at 37 degrees C. However, it is likely that only a fraction of the translocator rotates, the remainder being immobile over the measurement time of 500 microseconds. The mobile fraction of the translocator decreased with decrease in temperature. The observed fluorescence anisotropy of 0.24 indicates that EMA undergoes subnanosecond rapid wobbling in the binding site of the ADP/ATP translocator.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.     

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers

The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.     

Fatty acid-promoted mitochondrial permeability transition by membrane depolarization and binding to the ADP/ATP carrier

The mechanism by which non-esterified long-chain fatty acids (FFA) promote mitochondrial permeability transition (MPT) is not clear. We examined with energized rat liver mitochondria the role of two possible actions of FFA in MPT, (i) the reduction of the transmembrane potential (Δψ) and (ii) the increase of the negative surface charge of the inner mitochondrial membrane [Broekemeier, K.M. and Pfeiffer, D.G., Biochemistry 43, (1995) 16440–16449]. It was found that the ability of FFA to stimulate large amplitude swelling is clearly related to their uncoupling activity. Moreover, compared with classical protonophores (FCCP) FFA increase the sensitivity of the pore opening process to Δψ changes. In addition, FFA interact like their thioester derivatives in a structure-dependent manner with the ADP/ATP carrier (measured as inhibition of [³H]atractyloside binding to the AAC protein). It is suggested that not only the protonophoric action of FFA, but also a presumable stabilization of the ‘cytosolic' conformation of AAC contribute to the FFA-promoted MPT.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein.     

Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.     

Suggested mitochondrial ancestry of nonmitochondrial ATP/ADP carrier

One of the major evolutionary events that transformed an endosymbiotic bacterium into a mitochondrion was the acquisition of the ATP/ADP carrier (AAC) in order to supply the host with respiration-derived ATP. Along with the mitochondrial carrier, an unrelated carrier is known, which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic α-proteobacteria. This nonmitochondrial carrier was recently described in rickettsia-like endosymbionts (RLE), a group of obligate intracellular bacteria classified with the order Rickettsiales, which have diverged after free-living α-proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on nonmitochondrial AAC were re-analyzed in the present work, using both DNA and protein sequences and various methods including Bayesian analysis. The data presented are consistent with the classic endosymbiont theory for the origin of mitochondria and suggest that even the last but one common ancestor of rickettsiae and organelles was an endosymbiotic bacterium, in which AAC first originated.     

Interdependence between mitochondrial matrix volume and the extramitochondrial ratio of [ATP]/[ADP] [inorganic phosphate].

ATP or combinations of ATP with EDTA and EGTA can act as chelators to support succinate-driven, phosphate-requiring expansion of mitochondrial inner membrane-matrices. Contraction of these swollen mitochondria can be induced with antimycin, MgCl₂ and ADP. The magnitude of ADP-induced contraction of mitochondria, swollen in the presence of ATP, is dependent on [ADP] and may be altered by the extramitochondrial concentrations of both P_i and ATP. In fact, the extent of contraction (+ΔA₅₂₀) is a linear function of the thermodynamic parameter, ?ΔGp (free energy of hydrolysis of ATP), provided excessive concentrations of reactants are not present and the extents of matrix swelling are similar ($\text{e.g.}\text{ΔA}{}_{520}$ is about 0.250) before starting contraction with ADP.     

The biosynthesis of the mitochondrial ADP, ATP translocator.

The biosynthesis of the ADP,ATP carrier was studied in mitochondria of Neurospora crassa. The carrier was isolated as the carboxyatractylate-protein complex and characterized in dodecylsulphate/polyacrylamide gel electrophoresis to have a Mr = 33 000. Applying the inhibitors chloramphenicol for the intramitochondrial translation and cycloheximide for extramitochondrial translation, the site of synthesis of this polypeptide was found to be extramitochondrially located.     

Control of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate + rotenone and glutamate + malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized.The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.     

Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa.

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.