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At least 667 records · Page 37Linked to original sources
PubMed2006

Mdm38 interacts with ribosomes and is a component of the mitochondrial protein export machinery.

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologues of human Letm1, a protein implicated in Wolf-Hirschhorn syndrome. We analyzed the function of Mdm38 and Ylh47 in yeast mitochondria to gain insight into the role of Letm1. We find that mdm38Delta mitochondria have reduced amounts of certain mitochondrially encoded proteins and low levels of complex III and IV and accumulate unassembled Atp6 of complex V of the respiratory chain. Mdm38 is especially required for efficient transport of Atp6 and cytochrome b across the inner membrane, whereas Ylh47 plays a minor role in this process. Both Mdm38 and Ylh47 form stable complexes with mitochondrial ribosomes, similar to what has been reported for Oxa1, a central component of the mitochondrial export machinery. Our results indicate that Mdm38 functions as a component of an Oxa1-independent insertion machinery in the inner membrane and that Mdm38 plays a critical role in the biogenesis of the respiratory chain by coupling ribosome function to protein transport across the inner membrane.

Calcium-Binding Proteins↗
PubMed1992

Inhibition of hemin-induced hemolysis by desferrioxamine: binding of hemin to red cell membranes and the effects of alteration of membrane sulfhydryl groups.

Hemin binds to red cell membranes during hemin-induced hemolysis but the precise mechanism of hemolysis has not been characterized. Desferrioxamine (DFO), an iron chelator, inhibited hemin-induced hemolysis. DFO partially prevented hemin binding to red cell membranes and partially removed previously bound hemin. Glutathione, an intracellular sulfhydryl compound, also inhibited hemin-induced hemolysis but was only about one tenth as potent as DFO. Decrease of membrane sulfhydryl groups by treatment of cells with either N-ethylmaleimide (NEM) or diamide (azodicarboxylic acid bis [dimethylamide]) enhanced hemin-induced hemolysis. Enhancement of hemin-induced hemolysis by NEM and diamide and inhibition of hemolysis by DFO were independent with no evidence of synergism or interference between the two processes. Red cell membranes were saturated with hemin at approximately 75 nmol per mg protein. DFO decreased the hemin saturation level to 25 nmol per mg protein. In the presence of DFO, hemin was bound as the DFO-hemin complex since membranes preferentially removed DFO-hemin complexes from mixtures of complexed and free hemin while free DFO was not bound by the membranes. Access to the inner surface of the membrane was required for binding of the DFO-hemin complex since DFO completely prevented hemin binding in intact cells but not in cells undergoing hemolysis or red cell ghosts. Approximately 50 x 10(6) molecules of hemin were bound to the membrane of one red cell following hemin-induced hemolysis.

Deferoxamine↗
PubMed1982

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts.

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes.

Journal Article↗
PubMed2000

Mechanism of protein import across the chloroplast envelope.

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

Amino Acid Sequence↗
PubMed1978

Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis.

Isolated membrane fractions of Escherichia coli K-12 yielded complex immunoprecipitate patterns when Triton X-100 and sodium dodecyl sulfate extracts were examined by crossed immunoelectrophoresis with antienvelope immunoglobulins. Twelve of the 46 antigens in the immunoprecipitate patterns of inner (plasma) membranes were identified by zymograms and/or by the use of specific antisera. The following enzyme activities were detected in immunoprecipitates: 6-phosphogluconate dehydrogenase (EC 1.1.1.43); adenosine triphosphatase (EC 3.6.1.3); glutamate dehydrogenase (EC 1.4.1.4), two separate components; malate dehydrogenase (EC 1.1.1.37); dihydroorotate dehydrogenase (EC 1.3.3.1); succinate dehydrogenase (EC 1.3.99.1); lactate dehydrogeanse (EC 1.1.1.27); reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3); protease (EC 3.4.21.1); and glycerol 3-phosphate dehydrogenase (EC 1.1.99.5). The corresponding immunoprecipitate pattern for isolated outer membranes consisted of at least 25 discrete antigens and differed strikingly from that obtained with inner membranes. Two major immunogens were identified as lipopolysaccharide and Braun lipoprotein. A protease-active immunoprecipitate was also detected in this fraction, but attempts to identify the Rosenbusch matrix protein in the crossed immunoelectrophoretic profile were unsuccessful.

Antigens, Bacterial↗
PubMed1989

Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex.

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.

Adenosine Triphosphatases↗
PubMed1992

Anion permeation limits the uncoupling activity of fatty acids in mitochondria.

The action of such membrane-permeant cations as tetraphenyl phosphonium and dibenzyldimethyl ammonium upon fatty acid-uncoupled respiration has been studied with oligomycin-inhibited rat liver mitochondria. Both cations enhance fatty acid-stimulated respiration. This synergistic effect is explained by a facilitated permeation of the fatty acid anion across the inner membrane due to an ion-pair complex. It is concluded that fatty acid uncoupling in rat liver mitochondria is limited by fatty acid anion permeation.

2,4-Dinitrophenol↗
PubMed1995

Relocation during incubation of endothelial nuclei in the chick chorioallantois.

Early in incubation the nuclei of the chick chorioallantoic capillaries are randomly distributed around the capillary lumen; later most of them are located on the portion of the capillary surface opposite the inner shell membrane. This is one of the complex of processes that results in progressive thinning of the diffusion pathway for gases between the external environment and the blood of the embryo. The present study quantified this nuclear "relocation". Our data show a progressive relocation of the endothelial nuclei from the tenth through the sixteenth day at an average rate of 6% per day.

Age Factors↗
PubMed2006

Nuclear lamins, diseases and aging.

Nuclear lamins are type V intermediate filament proteins. They are the major building blocks of the peripheral nuclear lamina, a complex meshwork of proteins underlying the inner nuclear membrane. In addition to providing nuclear shape and mechanical stability, they are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly and nuclear positioning. Over the past few years, interest in the lamins has increased because of the identification of at least 12 distinct human diseases associated with mutations in the LMNA gene, which encodes A-type lamins. These diseases, collectively termed laminopathies, affect muscle, adipose, bone, nerve and skin cells and range from muscular dystrophies to accelerated aging.

Aging↗
PubMed1999

Chloroplast precursor protein translocon.

Chloroplasts are believed to have originated from a photosynthetic, prokaryotic ancestor. As the result of endosymbiotic evolution, most of the genes of the endocytobiont were displaced to the host nucleus. Today's chloroplasts must import most of their proteins from the cytosol as precursors. Oligomeric protein complexes in the chloroplast outer and inner envelope membranes are responsible for the specific recognition and membrane translocation of precursor proteins. The translocon at the outer membrane of chloroplasts and the inner membrane of chloroplasts act jointly during the import process. Several translocon subunits have been partially characterized in their molecular structure and function. Initial evidence indicates the prokaryotic origin of some chloroplast translocon components.

Biological Transport↗
PubMed2000

The internal structure of mitochondria.

Electron microscopic (EM) tomography is providing important new insights into the internal organization of mitochondria. The standard baffle model for cristae structure, called into question years ago, has now clearly been shown to be inaccurate. Depending on source and conformational state, cristae can vary from simple tubular structures to more complex lamellar structures merging with the inner boundary membrane through tubular structures 28 nm in diameter. The structural information provided by EM tomography has important implications for mitochondrial bioenergetics, biogenesis and the role of mitochondria in apoptosis. The structural paradigm defined by EM tomography is helping in the design of new experimental approaches to mitochondrial function.

Animals↗
PubMed1989

Nitroreductase activity of NADH dehydrogenase of the respiratory redox chain.

1. An NADH-dependent nitroreductase from the inner membrane of ox liver mitochondria copurified with Complex I of the respiratory redox chain (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). 2. The corresponding nitroreductase from ox heart mitochondria co-purified with the NADH-cytochrome c reductase of Mahler, Sarkar & Vernon [(1952) J. Biol. Chem. 199, 585-597] [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], a component of Complex I that contains the FMN. 3. The mitochondrial nitroreductase activity is attributed to the flavoprotein component of Complex I.

Animals↗
PubMed2003

SecYEG proteoliposomes catalyze the Deltaphi-dependent membrane insertion of FtsQ.

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particle-dependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.

Adenosine Triphosphatases↗
PubMed1998

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

Adenosine Triphosphatases↗
PubMed2001

Intracellular induction of the Bartonella henselae virB operon by human endothelial cells.

One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2 gene revealed a region with sequence homology to the vir box of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virB genes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A. tumefaciens.

Bacterial Proteins↗
PubMed1999

Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii.

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.

Amino Acid Sequence↗
PubMed2000

Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems.

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

Bacterial Proteins↗
PubMed2006

Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

Animals↗