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PubMed2006

Preliminary X-ray crystallographic studies of yeast mitochondrial protein Tom70p.

Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tom70p is an important TOM-complex member and a major surface receptor of the protein-translocation machinery in the outer mitochondrial membrane. To investigate the mechanism by which Tom70p functions to deliver the mitochondrial protein precursors, the cytosolic fragment of yeast Tom70p (cTom70p) was crystallized. The crystals diffract to 3.2 A using a synchrotron X-ray source and belong to space group P2(1), with unit-cell parameters a = 44.89, b = 168.78, c = 83.41 A, alpha = 90.00, beta = 102.74, gamma = 90.00 degrees. There are two Tom70p molecules in one asymmetric unit, which corresponds to a solvent content of approximately 51%. Structure determination by MAD methods is under way.

Crystallization↗
PubMed2005

Role of Tim21 in mitochondrial translocation contact sites.

Translocation of preproteins with N-terminal presequences into mitochondria requires the cooperation of the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). However, the molecular nature of the translocation contact sites is poorly understood. We have identified a novel component of the TIM23 translocase, Tim21, which is involved in their formation. Tim21 is anchored in the mitochondrial inner membrane by a single transmembrane domain and exposes its C-terminal domain into the intermembrane space. The purified C-terminal domain of Tim21 appears not to bind to any of the TIM23 components but rather specifically interacts with the TOM complex. We propose that Tim21 binds to the trans site of the TOM complex thus keeping the two translocases in close contact.

Amino Acid Sequence↗
PubMed2001

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1.

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.

Acetylation↗
PubMed1985

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex.

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.

Animals↗
PubMed2000

Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins.

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

Amino Acid Sequence↗
PubMed2005

Detection and identification of stable oligomeric protein complexes in Escherichi coli inner membranes: a proteomics approach.

In this study we present a new technology to detect stable oligomeric protein complexes in membranes. The technology is based on the ability of small membrane-active alcohols to dissociate the highly stable homotetrameric potassium channel KcsA. It is shown via a proteomics approach, using diagonal electrophoresis and nano-flow liquid chromatography coupled to tandem mass spectrometry, that a large number of both integral and peripheral Escherichia coli inner membrane proteins are part of stable oligomeric complexes that can be dissociated by small alcohols. This study gives insight into the composition and stability of these complexes.

Bacterial Proteins↗
PubMed1988

Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma.

Gram-negative bacteria are surrounded by two membranes. In these bacteria, a class of high affinity transport systems for concentrating substrates from the medium into the cell, involves a binding protein located between the outer and inner membranes, in the periplasmic region. These 'periplasmic binding-proteins' are thought to bind the substrate in the vicinity of the inner membrane, and to transfer it to a complex of inner membrane proteins for concentration into the cytoplasm. We report evidence leading us to propose that a Gram-positive bacterium, Streptococcus pneumoniae, and a mycoplasma, Mycoplasma hyorhinis, which are surrounded by a single membrane and have therefore no periplasmic region, possess an equivalent to the high affinity periplasmic binding-protein dependent transport systems, i.e. extra-cytoplasmic binding lipoprotein dependent transport systems. The 'binding lipoproteins' would be maintained at proximity of the inner membrane by insertion of their N-terminal glyceride-cysteine into this membrane.

Amino Acid Sequence↗
PubMed2003

Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex.

Preproteins with N-terminal presequences are imported into mitochondria at translocation contact sites that include the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Little is known about the functional cooperation of these translocases. We have characterized translocation contact sites by a productive TOM-TIM-preprotein supercomplex to address the role of three translocase subunits that expose domains to the intermembrane space (IMS). The IMS domain of the receptor Tom22 is required for stabilization of the translocation contact site supercomplex. Surprisingly, the N-terminal segment of the channel Tim23, which tethers the TIM23 complex to the outer membrane, is dispensable for both protein import and generation of the TOM-TIM supercomplex. Tim50, with its large IMS domain, is crucial for generation but not for stabilization of the supercomplex. Thus, Tim50 functions as a dynamic factor and the IMS domain of Tom22 represents a stabilizing element in formation of a productive translocation contact site supercomplex.

Carrier Proteins↗
PubMed2005

Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.

Amino Acid Sequence↗
PubMed1998

Import of mitochondrial carriers mediated by essential proteins of the intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

Biological Transport↗
PubMed1978

Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli.

DNA-envelope complexes isolated from osmotically lysed spheroplasts of Escherichia coli contained 0.2 to 1% of the total cellular DNA after labeling with [3H]thymidine. Molecular weight determinations indicated that the amount of bound DNA was equivalent in most cases to a maximum of three binding sites per chromosome. Bound DNA from E. coli B/r was distributed approximately equally between inner and outer membrane components when envelopes were fractionated on sucrose equilibrium gradients. Outer membrane-DNA complexes, in particular, fraction H1, with a density of 1.24 g/cm3, were quite stable against shearing and against Sarkosyl NL97. In the case of E. coli B/r, H1-DNA was also relatively resistant to deoxyribonuclease. Inner membrane-DNA complexes, in contrast, were quite labile and readily dissociated to release free DNA. The outer membrane fractions did not appear to contain replication fork DNA, but small amounts may have been present in the inner membrane complexes. A two- to eightfold enrichment for chromosomal origin DNA in the envelope was obtained when cultures of E. coli K-12, synchronized for DNA replication, were pulse labeled at different times in the replication cycle. This enrichment was found invariably in the outer membrane fractions. However, the data do not exclude the possibility that this DNA is bound to regions of adhesion between inner and outer membranes which sediment with a density indistinguishable from that of the outer membrane.

Binding Sites↗
PubMed1994

On the interactions of Ca2+ and cyclosporin A with a mitochondrial inner membrane pore: a study using cobaltammine complex inhibitors of the Ca2+ uniporter.

The mitochondrial inner membrane contains a Ca(2+)-activated pore of possible relevance to the pathogenesis of ischaemia/reperfusion injury which is inhibited by the immunosuppressant cyclosporin A (CSA). The present study employs a number of novel cobaltammine complex inhibitors of the Ca2+ uniporter (mediating Ca2+ uptake) to examine whether intramitochondrial Ca2+ influences the capacity of CSA to block the pore. Using dissipation of the inner membrane potential as a means of monitoring the state of the pore, it is shown that CSA blockade is facilitated as Ca2+ uptake is restricted. Ca2+ also depresses and reverses the binding of [3H]CSA to mitochondria, but Ca2+ is ineffective when its uptake is prevented. It is concluded that a high intramitochondrial Ca2+ concentration antagonizes pore inhibition by CSA. The significance of this is discussed.

Animals↗
PubMed1998

Evidence of a subunit 4 (subunit b) dimer in favor of the proximity of ATP synthase complexes in yeast inner mitochondrial membrane.

Yeast mitochondria having either the D54C or E55C mutations in subunit 4 (subunit b), which is a component of the ATP synthase stator, displayed a spontaneous disulfide bridge between two subunits 4. This dimer was not soluble upon Triton X-100 extraction either at concentrations which extract the yeast ATP synthase or at higher concentrations. Increasing detergent concentrations led to a lack of the oligomycin-sensitive ATPase activity, thus showing an uncoupling between the two sectors of the mutated enzymes due to the dissociation of the subunit 4 dimer from the mutant enzyme. There is only one subunit 4 (subunit b) per eukaryotic ATP synthase. As a consequence, the results are interpreted as the proximity of ATP synthase complexes within the inner mitochondrial membrane.

Intracellular Membranes↗
PubMed1989

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane.

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.

Antibodies, Bacterial↗
PubMed1993

Polar location of the chemoreceptor complex in the Escherichia coli cell.

The eukaryotic cell exhibits compartmentalization of functions to various membrane-bound organelles and to specific domains within each membrane. The spatial distribution of the membrane chemoreceptors and associated cytoplasmic chemotaxis proteins in Escherichia coli were examined as a prototypic functional aggregate in bacterial cells. Bacterial chemotaxis involves a phospho-relay system brought about by ligand association with a membrane receptor, culminating in a switch in the direction of flagellar rotation. The transduction of the chemotaxis signal is initiated by a chemoreceptor-CheW-CheA ternary complex at the inner membrane. These ternary complexes aggregate predominantly at the cell poles. Polar localization of the cytoplasmic CheA and CheW proteins is dependent on membrane-bound chemoreceptor. Chemoreceptors are not confined to the cell poles in strains lacking both CheA and CheW. The chemoreceptor-CheW binary complex is polarly localized in the absence of CheA, whereas the chemoreceptor-CheA binary complex is not confined to the cell poles in strains lacking CheW. The subcellular localization of the chemotaxis proteins may reflect a general mechanism by which the bacterial cell sequesters different regions of the cell for specialized functions.

ATP-Binding Cassette Transporters↗
PubMed2005

Biogenesis of cytochrome oxidase-sophisticated assembly lines in the mitochondrial inner membrane.

Biogenesis of the cytochrome oxidase complex in the mitochondrial inner membrane depends on the concerted action of a variety of proteins. Recent studies shed light on this biological assembly process revealing an astonishingly complex procedure by which the different subunits of the enzymes are put together and the required cofactors are supplied. In this review we present a hypothetical model for the assembly process of cytochrome oxidase based on the current knowledge of the functions of specific assembly factors. According to this model the two largest subunits of the complex are first equipped with their respective cofactors on independent assembly lines. Prior to their assembly with the residual subunits that complete the whole complex, these two subcomplexes remain bound to substrate-specific chaperones. We propose that these chaperones, Mss51 for subunit 1 and Cox20 for subunit 2, control the coordinate assembly process to prevent potentially harmful redox reactions of unassembled or misassembled subunits.

Amino Acid Sequence↗
PubMed2005

Nuclear envelopathies--raising the nuclear veil.

The nuclear envelope separates the chromosomes from cytoplasm in eukaryotic cells and consists of three main domains: inner and outer nuclear membranes and nuclear pore complexes. The inner nuclear membrane maintains close associations with the underlying chromatin and nuclear lamina. For many years, the nuclear envelope was thought to function mainly as an architectural stabilizer of the nucleus, participating in assembly and disassembly processes during mitosis. However, recent findings demonstrate that nuclear envelope proteins are involved in fundamental nuclear functions, such as gene transcription and DNA replication, and that inherited or de novo mutated proteins cause human diseases, termed "nuclear envelopathies." These findings emphasize the importance of understanding the functions of this cellular domain, in both physiologic and pathologic states. To date, mutations in the genes encoding the nuclear envelope proteins emerin, MAN1, lamin A/C, and lamin B receptor were found to cause nuclear envelopathies. The diseases that are caused by mutations in LMNA gene are collectively called "laminopathies." Nuclear envelopathies have diverse clinical phenotypes, ranging from cardiac and skeletal myopathies to partial lipodystrophy, peripheral neuropathy, and premature aging. This raises the question of how do such ubiquitously expressed proteins give rise to tissue-specific disease phenotypes. One possible explanation is the involvement of nuclear envelope proteins in the regulation of gene transcription, a novel mechanism that has been the focus of research in our lab in recent years. In this review, we describe recent discoveries in the field of nuclear envelopathies and discuss current proposed pathophysiological mechanisms underlying these diseases.

Animals↗
PubMed1979

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.

Animals↗