Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane.

In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl-p-phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (pK = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline pK, aspartate efflux is inhibited with increased matrix [H⁺] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H⁺]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δp) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H⁺] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H⁺] and was unaffected by the energy state.The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 mm, however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1–35 °C and 5–25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1–2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed.     

Protein transport machineries for precursor translocation across the inner mitochondrial membrane

The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.     

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane.

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex. (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.     

The effect of adriamycin and duramycin on calcium translocation in liposome systems modeled on the inner mitochondrial membrane

Adriamycin (doxorubicin, AdM) is a potent antineoplastic agent which binds specifically and with high affinity to the acidic phospholipid cardiolipin (CL) [Goormaghtigh et al. (1980) Biochim. Biophys. Acta 597, 1]. Duramycin (DM), a polypeptide antibiotic, has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol [Navarro et al. (1985) Biochemistry 24, 4645]. The selectivity of DM-PE interaction was confirmed. AdM and DM were then used to explore the roles of CL and PE in Ca2+ translocation in a phosphatidylcholine (PC)/PE/CL liposome system modeled on the inner mitochondrial membrane with the following results: (i) AdM (100-400 microM) altered Ca2+ uptake by PC/PE/CL (4/4/1, mol/mol) liposomes in a concentration-dependent fashion which varied with temperature, external Ca2+ concentration, and liposome PE content. (ii) Addition of AdM was qualitatively equivalent to increasing temperature, Ca2+ concentration, or liposome PE content, and cooperative interactions among these parameters were observed. An increase in any one factor generally enhanced Ca2+ uptake; simultaneous increases in several factors inhibited uptake. (iii) Inhibition of Ca2+ uptake was correlated with efflux of Arsenazo III. (iv) Ca2+ uptake by PC/PE/CL liposomes is biphasic [Kester and Sokolove (1989) Biochim. Biophys. Acta 980, 127]. DM suppressed the PE-dependent slow phase and stimulated the PE-independent initial phase. Ca2+ uptake by PC/PE/CL liposomes in the presence of DM resembled uptake by PC/CL liposomes. These data confirm the ability of PE to enhance the slow, highly temperature-dependent component of CL-mediated Ca2+ translocation and suggest that this process is sensitive to lipid phase behavior.     

Calcium ion activation of the anion-conducting channel in the rat liver mitochondrial inner membrane.

Stimulation of the rat liver mitochondrial inner-membrane anion-conducting channel by aeration is dependent on the concentration of Ca2+ ions in the assay medium. Ca2+ activates anion conduction in both aerated and non-energised mitochondria but acts over a wider concentration range and produces a greater increase in anion-conductivity in aerated mitochondria. EGTA reverses Ca2+ stimulation but takes several seconds to act, indicating slow release of Ca2+ from the activation site possibly on the matrix side of the inner mitochondrial membrane. It is suggested that this channel may respond to hormone-induced changes in cytosolic Ca2+ concentration.     

Dynamic subcompartmentalization of the mitochondrial inner membrane

The inner membrane of mitochondria is organized in two morphologically distinct domains, the inner boundary membrane (IBM) and the cristae membrane (CM), which are connected by narrow, tubular cristae junctions. The protein composition of these domains, their dynamics, and their biogenesis and maintenance are poorly understood at the molecular level. We have used quantitative immunoelectron microscopy to determine the distribution of a collection of representative proteins in yeast mitochondria belonging to seven major processes: oxidative phosphorylation, protein translocation, metabolite exchange, mitochondrial morphology, protein translation, iron-sulfur biogenesis, and protein degradation. We show that proteins are distributed in an uneven, yet not exclusive, manner between IBM and CM. The individual distributions reflect the physiological functions of proteins. Moreover, proteins can redistribute between the domains upon changes of the physiological state of the cell. Impairing assembly of complex III affects the distribution of partially assembled subunits. We propose a model for the generation of this dynamic subcompartmentalization of the mitochondrial inner membrane.     

Reducibility of cytochromeb in mitochondrial inner membrane

When mitochondrial inner membrane was disintegrated into Complex I-III, IV, and oligomycin-sensitive ATPase, about 50% of cytochromeb in Complex I-III was readily reduced with NADH, as judged by the appearance of a peak at 562 nm, while in whole mitochondria less than 25% of cytochromeb was reduced by succinate. On addition of antimycin to the substrate-reduced Complex I-III, cytochromeb was further reduced to 71% of the total, and the peak at 562 nm was red-shifted to 564 nm as in the case of dithionite reduction. These results indicate that the 562 nm and 564 nm peaks, at 29°C correspond, respectively, tob ₅₆₀ andb _562.5 at 77°K of Davis et al. [7] and to b _K and b _T of Chance et al. [2]. When Complex I-III and oligomycin-sensitive ATPase were reconstituted to form a membrane, about 60% of cytochromeb in Complex I-III was readily reduced with NADH. In this case the 562 nm peak was not red-shifted. However, the difference spectrum of NADH-reduced membraneminus that in the presence of deoxycholate showed a peak at 565 nm. A mirror image of the difference spectrum was obtained on addition of an uncoupler,m-chlorocarbonyl cyanide phenylhydrazone. This is characteristic for b _T. These results support the idea that the occurrence of spectral peaks of b _T and b _K is not due to two species but to single speciesAbbreviations OS-ATPase oligomycin sensitive ATPase - CCCP m-chlorocarbonyl cyanide phenylhydrazone - F₁ coupling factor one - OSCP oligomycinsensitivity-conferring protein