Proteins required for the binding of mitrochondrial ATPase to the mitochondrial inner membrane.

1. Isolated F1 (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 muM. 2. About one-third of the F1 and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles). 3. After further treatment with alkali of urea-treated particles, binding of F1 requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc2 are added. The concentration of F1-binding sites in the presence of both OSCP and Fc2 is about the same as that in TU particles. 4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F1, unless Fc2 is also present. Fc2 induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive. 5. It is tentatively concluded that OSCP is the binding site for F1 and Fc2 is the binding site for OSCP.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

35S-Atractyloside binding affinity to the inner mitochondrial membrane

Isolated inner mitochondrial membrane contains a small number of binding sites for atractyloside (of the order of 0.1 nmole/mg of protein) with very high binding affinity (half saturation at 0.014 &mgr;M atractyloside). The high affinity binding ability of the inner mitochondrial membrane is markedly decreased upon aging, acidification of the medium or addition of ADP, but remains unchanged in the presence of uncouplers such as FCCP. Added ADP causes a two-step transition from the high affinity binding to low affinity binding (K(d) > 0.50 &mgr;M) and concomitantly a significant increase of the measured number of binding sites (about a doubling). The half maximum effect in the first step transition is given by 1 &mgr;M ADP. The use of 35S-atractyloside as a probe of the inner mitochondrial membrane conformation specifically related to the adenine nucleotide translocation is discussed.     

On the relationship between mitochondrial ATPase and the inner membrane

Digestion of the submitochondrial particle (ETP_H) with a proteolytic enzyme, Nagarse, selectively and completely removed the headpieces from the membrane without damaging the electron transfer chain. By determining the amount of protein released by the Nagarse treatment, it was calculated that the headpieces represent 16±0.5% of the total protein of the submitochondrial particles.In respiring ETP_H, membrane-bound AMP was found to be an acceptor of inorganic phosphate, and this esterification led to the formation of membrane-bound ADP. About 70% of the membranebound adenine nucleotides were found to be tightly bound to the intrinsic proteins of the membrane. A transphosphorylation reaction was observed between external and membrane-bound ADP.Abbreviations F₁ coupling factor one - OSCP oligomycin-sensitivity conferring protein - TRU tripartite repeating unit - ETP_H phosphorylating electron transfer particle     

Photodynamic action of bilirubin on the inner mitochondrial membrane. Implications for the organization of the mitochondrial ATPase

Bilirubin in the presence of O₂ and light catalyzes the photodynamic modification of the proteins of the inner mitochondrial membrane as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Numerous polypeptide bands become streaked towards higher apparent molecular weight and decrease in staining intensity while other bands remain largely unchanged. The loss in staining intensity which occurs is at least partially due to apparent cross-linking of the polypeptides to produce aggregates which cannot penetrate into the gel. The α and β bands of the mitochondrial ATPase differ markedly in their susceptibility to modification. The β subunit is rapidly modified while the α subunit is largely inert. This differential susceptibility is a consequence of the binding of the soluble F₁ ATPase to the membrane. When submitochondrial particles with their normal complement of bound F₁ are mixed with free F₁ and are photolyzed together in the presence of bilirubin and O₂, it is found that the β subunit of the membrane-bound F₁, but not the α subunit, has been modified while neither subunit of the free F₁ has been modified. This increased susceptibility of the β subunit in the membrane state may represent cross-linking to membrane components and is consistent with the β subunit making more extensive contacts with membrane components than does the α subunit.     

A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane

A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.     

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.     

Electrophysiology of the inner mitochondrial membrane

The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K⁺-selective one, a high-conductance megachannel, a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca²⁺ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies.     

An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth.     

Pathways shaping the mitochondrial inner membrane

Mitochondria are complex organelles with two membranes. Their architecture is determined by characteristic folds of the inner membrane, termed cristae. Recent studies in yeast and other organisms led to the identification of four major pathways that cooperate to shape cristae membranes. These include dimer formation of the mitochondrial ATP synthase, assembly of the mitochondrial contact site and cristae organizing system (MICOS), inner membrane remodelling by a dynamin-related GTPase (Mgm1/OPA1), and modulation of the mitochondrial lipid composition. In this review, we describe the function of the evolutionarily conserved machineries involved in mitochondrial cristae biogenesis with a focus on yeast and present current models to explain how their coordinated activities establish mitochondrial membrane architecture.     

Gluing the respiratory chain together. Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane

Cytochrome bc(1) complex (complex III) and cytochrome c oxidase complex (complex IV) are multisubunit homodimers that are essential components of the mitochondrial respiratory chain. Complexes III and IV associate to form a supercomplex that can be displayed using blue native polyacrylamide gel electrophoresis. Both homodimeric complexes contain tightly associated cardiolipin (CL) required for function. We report here that in a crd1Delta strain of yeast (null in expression of CL synthase) approximately 90% of complexes III and IV were observed as individual homodimers; only the supercomplex was observed with CRD1 wild type cells. Introduction of a plasmid born copy of the CRD1 gene under exogenous regulation by doxycycline made possible controlled variation in the in vivo CL levels. At an intermediate level of CL, a mixture of individual homodimers (30%) and supercomplex (70%) was observed. These results strongly indicate that CL plays a central role in higher order organization of components of the respiratory chain of mitochondria.     

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.     

Protein crowding in the inner mitochondrial membrane

The inner membrane of mitochondria is known for its low lipid-to-protein ratio. Calculations based on the size and the concentration of the principal membrane components, suggest about half of the hydrophobic volume of the membrane is occupied by proteins. Such high degree of crowding is expected to strain the hydrophobic coupling between proteins and lipids unless stabilizing mechanisms are in place. Both protein supercomplexes and cardiolipin are likely to be critical for the integrity of the inner mitochondrial membrane because they reduce the energy penalty of crowding.