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At least 433 records · Page 24Linked to original sources
PubMed1995

A two cell type theory for aldosterone biosynthesis: the roles of 11 beta-hydroxylase and aldosterone synthase, and a high capacity tightly binding steroid carrier for 18-hydroxydeoxycorticosterone in rat adrenals.

Several lines of experimentation suggest that a tissue-sequestered pool of 18-hydroxydeoxycorticosterone (18-OH-DOC) in the rat adrenal may be mobilized as an aldosterone precursor. We show here that this steroid is maintained in a non-extractable form in the membranes of collagenase-dispersed fasciculata/reticularis cells. Because of this stability, the complex can be identified by immunocytochemistry and also, in IEF gels of solubilized inner adrenocortical zone membrane preparations, by immunoblotting. However, the complexed steroid cannot be extracted from the gels into organic solvent unless first treated with trypsin. Preincubation of viable whole glandular tissue with trypsin significantly enhanced aldosterone output and eliminated the trypsin-releasable 18-OH-DOC pool in IEF gels of solubilized inner zone membranes. Both prior sodium depletion and acute trypsin stimulation of whole glands enhanced extractable 18-OH-DOC in glomerulosa tissue membranes. Other experiments using in situ hybridization show that mRNA coding for 11 beta-hydroxylase (which generates 18-OH-DOC) is confined to the inner adrenocortical zones, whereas aldosterone synthase (which does not) is transcribed exclusively in the glomerulosa. The data suggest that a pool of 18-OH-DOC in inner zone membranes can be mobilized for utilization as an aldosterone precursor in the glomerulosa. The results also indicate the existence of an entirely novel tightly binding steroid carrier from which steroid cannot be extracted by organic solvent unless first subjected to proteolytic degradation.

18-Hydroxydesoxycorticosterone↗
PubMed2006

Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria.

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

Fungal Proteins↗
PubMed2002

The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease.

Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phblp and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phblp and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately.

Aging↗
PubMed2003

The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) interacts with mitochondrial bioenergetic functions.

The effects of dicamba, a widely used broad-leaf herbicide, on rat liver mitochondrial bioenergetic activities were examined. The results obtained for state 4 respiration indicate not only an uncoupling effect, the result of an increase on the permeability of inner mitochondria membrane to protons, but also a strong inhibitory effect on the redox complexes. State 3 and respiration uncoupled by FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) were inhibited to approximately the same extent, i.e. by about 70%. Depression of respiratory activity is essentially mediated through partial inhibition of mitochondrial complexes II and III. ATPase activity was much less depressed by dicamba than ATP synthase activity. Therefore, a considerable part of the inhibition observed on ATP synthase is the result of an inhibition on the redox complexes. The loss of phosphorylation capacity, induced by dicamba, was in the last analysis not only the result of a direct effect of dicamba on the enzymatic complex (F(0)-F(1) ATPase) but also the result of a deleterious effect on the integrity of the mitochondrial membrane, which can promote an inhibition of the respiratory complexes and an increase of the proton permeability of the inner membrane.

Animals↗
PubMed1999

Oxidative phosphorylation at the fin de siècle.

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5'-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here.

Adenosine Triphosphate↗
PubMed2006

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE.

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.

ATP-Binding Cassette Transporters↗
PubMed2002

Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect.

The mechanism by which external Bax releases cytochrome c is still controversial and may also depend on the type of mitochondria and the actual localisation of cytochrome c. Outer membrane porin acquires high binding affinity for hexokinase by interacting with the adenine nucleotide translocator (ANT) in the contact sites. (I) The hexokinase protein was thus used as a tool to isolate the contact site forming complex between outer membrane porin and inner membrane ANT from a TritonX100 extract of brain membranes. (II) A significant amount of cytochrome c was co-purified with the isolated hexokinase porin ANT complexes that were reconstituted in phospholipid vesicles. Bax-AC released the endogenous cytochrome c from the vesicles without forming unspecific pores. This was shown by loading the vesicles with malate that was not liberated by Bax-AC. (III) The Bax-AC effect was dependent on a specific association of cytochrome c with the porin ANT complex, as dissociation of the complex by bongkrekate abolished the Bax dependent cytochrome c liberation. (IV) The Bax-AC effect was as well suppressed by hexokinase phosphorylating glucose.

Binding Sites↗
PubMed2004

Formation of membrane-bound ring complexes by prohibitins in mitochondria.

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of approximately 1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of approximately 120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of approximately 270 x 200 angstroms. Implications of these findings for proposed cellular activities of prohibitins are discussed.

Amino Acid Sequence↗
PubMed1979

Topological studies of cytochromes P-450scc and P-45011 beta in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion.

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria were studied by using controlled digestion with trypsin of purified inner mitochondrial membranes. Inhibition of steroid hydroxylase activity by trypsin was only observed in inner mitochondrial membranes which had been disrupted by various techniques. The steroid hydroxylase activity of intact inner membranes was not inhibited by trypsin. The effect of tryptic digestion was monitored by measuring 11 beta-hydroxylase and cholesterol side chain cleavage activities, as well as cytochrome P-450 reduction. The effect of trypsin on the steroid-induced difference spectra using pregnenolone, 20 alpha-hydroxycholesterol, and deoxycorticosterone was also measured. The results were similar regardless of which procedure was utilized and strongly suggest that both cytochrome P-45011 beta and cytochrome P-450scc are located on the matrix side of the mitochondrial inner membrane.

Adrenal Cortex↗
PubMed2004

AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex.

Many transporters of Gram-negative bacteria involved in the extracellular secretion of proteins and the efflux of toxic molecules operate by forming intermembrane complexes. These complexes are proposed to span both inner and outer membranes and create a bridge across the periplasm. In this study, we analyzed interactions between the inner and outer membrane components of the tri-partite multidrug efflux pump AcrAB-TolC from Escherichia coli. We found that, once assembled, the intermembrane AcrAB-TolC complex is stable during the separation of the inner and outer membranes and subsequent purification. All three components of the complex co-purify when the affinity tag is attached to either of the proteins suggesting bi-partite interactions between AcrA, AcrB, and TolC. We show that antibiotics, the substrates of AcrAB-TolC, stabilize interactions within the complex. However, the formation of the AcrAB-TolC complex does not require an input of energy.

Carrier Proteins↗
PubMed1997

Coupling chemical energy by the hsp70/tim44 complex to drive protein translocation into mitochondria.

A dynamic complex between the mitochondrial cognate of hsp70 (mthsp70) and the inner membrane protein tim44 couples energy derived from ATP hydrolysis to drive multiple steps in the mitochondrial protein import pathway: (1) The delta psi dependent import step and the mthsp70/tim44 complex cooperate to facilitate the unidirectional transfer of the mitochondrial targeting signal across the inner membrane. (2) The mthsp70/tim44 complex helps to unfold domains on precursors proteins that arrive at the import apparatus in a folded conformation on the cis side of the outer membrane. (3) Completion of import is then driven by the mthsp70/ tim44 complex in a manner that is independent of delta psi. Mechanisms proposed to explain how the mthsp70/tim44 complex harvests chemical energy to drive these aspects of the import process are discussed.

Carrier Proteins↗
PubMed2001

Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins.

The biogenesis of membrane oligomeric complexes is an intricate process that requires the insertion and assembly of transmembrane (TM) domains into the lipid bilayer. The Oxa1p family plays a key role in this process in organelles and bacteria. Hell et al. (2001, EMBO J., 20, 1281-1288) recently have proposed that Oxa1p could act as part of a general membrane insertion machinery for mitochondrial respiratory complex subunits. We have previously shown that mutations in the TM domain of Cyt1p can partially compensate for the absence of Oxa1p. Here, we demonstrate that a single amino acid substitution in the TM domain of Qcr9p can bypass Oxa1p in yeast. Qcr9p and Cyt1p are two subunits of the respiratory complex bc1 and their relative roles in the assembly of other respiratory complexes have been investigated. The mutations we have isolated in Cyt1p or Qcr9p introduce positively charged amino acids, and we show that the mutant TM domain of Cyt1p mediates the restoration of complex assembly. We propose that the positive charges introduced in Cyt1p and Qcr9p TM domains promote interactions with negatively charged TM domains of other respiratory complex subunits, allowing the coinsertion of both domains into the membrane, in the absence of Oxa1p. This model argues in favor of a role of Oxa1p in the insertion and the lateral exit of less hydrophobic TM domains from the translocation site into the lipid bilayer.

Amino Acid Substitution↗
PubMed2003

Maltose transport through the inner membrane of E. coli.

The maltose transport complex of E.coli is one of the most well-characterized members of the ATP-Binding Cassette (ABC) protein superfamily. ABC proteins represent the largest superfamily of transmembrane proteins in prokaryotes and eukaryotes, performing diverse functions from ion transport by the cystic fibrosis transmembrane regulator to multiple drug efflux by the P-glycoprotein transporter and sugar transport by the maltose transporter. Characterization of the mechanism of transport for ABC transporters is currently being investigated both biochemically and structurally, however some uncertainty remains as to how the individual subunits of these multisubunit transporters interact. This review discusses the current knowledge of the mechanism of maltose transport, as it relates to the ABC superfamily of transporters as a whole.

Amino Acid Sequence↗
PubMed1995

Primary and secondary carnitine deficiency syndromes.

The objective of this article is to review primary and secondary causes of carnitine deficiency, emphasizing recent advances in our knowledge of fatty acid oxidation. It is now understood that the cellular metabolism of fatty acids requires the cytosolic carnitine cycle and the mitochondrial beta-oxidation cycle. Carnitine is central to the translocation of the long chain acyl-CoAs across the inner mitochondrial membrane. The mitochondrial beta-oxidation cycle is composed of a newly described membrane-bound system and the classic matrix compartment system. Very long chain acyl-CoA dehydrogenase and the trifunctional enzyme complex are embedded in the inner mitochondrial membrane, and metabolize the long chain acyl-CoAs. The chain shortened acyl-CoAs are further degraded by the well-known system in the mitochondrial matrix. Numerous metabolic errors have been described in the two cycles of fatty acid oxidation; all are transmitted as autosomal recessive traits. Primary or secondary carnitine deficiency is present in all these clinical conditions except carnitine palmitoyltransferase type I and the classic adult form of carnitine palmitoyltransferase type II deficiency. The sole example of primary carnitine deficiency is the genetic defect involving the active transport across the plasmalemmal membrane. This condition responds dramatically to oral carnitine therapy. The secondary carnitine deficiencies respond less obviously to carnitine replacement. These conditions are managed by high carbohydrate, low fat frequent feedings, and vitamin/cofactor supplementation (eg, carnitine, glycine, and riboflavin). Medium chain triglycerides may be useful in the dietary management of patients with inborn errors of the cytosolic carnitine cycle or the mitochondrial membrane-bound long chain specific beta-oxidation system.

Animals↗
PubMed1995

Mitochondrial proton conductance and H+/O ratio are independent of electron transport rate in isolated hepatocytes.

In this paper we examine the non-linearity of the relationship between the proton electrochemical gradient across the mitochondrial inner membrane (delta p) and oxygen consumption of non-phosphorylating mitochondria in situ in hepatocytes. Models proposing to explain the non-linear relationship were tested experimentally. It was shown that the mitochondrial proton conductance and the number of protons pumped to the cytosolic side of the mitochondrial inner membrane by the electron transport complexes per oxygen atom consumed (H+/O ratio) are independent of electron transport rate in mitochondria in isolated hepatocytes. The non-linearity of the plot of delta p against the non-phosphorylating oxygen consumption is due to either a potential-dependent slippage of the proton pumps of the mitochondrial inner membrane and/or a potential-dependent leakage of protons back across the mitochondrial inner membrane.

Animals↗
PubMed1988

Influence of Ca2+ on the isolation from rat brain mitochondria of a fraction enriched of boundary membrane contact sites.

Data have been obtained suggesting that the complex porin-hexokinase of brain mitochondria may be related to the contact sites between the outer and inner membrane. In the attempt to isolate from brain mitochondria the inner and outer membranes and the boundary membrane contacts, a procedure was developed based on swelling and shrinking of the organelles, followed by sonication and reverse discontinuous density gradient centrifugation. Three fractions were obtained by this technique, which were identified by measuring the relative specific activities of marker enzymes, namely succinate-cytochrome c reductase; NADH-cytochrome c reductase (rotenone insensitive); hexokinase and glutathione transferase, for the inner and outer membranes and contact sites, respectively. The fraction which contains the contact sites is characterized by the highest specific activity of hexokinase and glutathione transferase and by the highest calcium binding capacity; physiological concentrations of this cation produces a sharper separation of this fraction. Results indicate that both the porin-hexokinase gating system of the outer membrane and the calcium transporting complex of the inner membrane are present in the fraction which contains the contact sites.

Animals↗
PubMed2003

Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae.

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5'-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system.

Cyclooxygenase 1↗
PubMed1998

Mitochondria in neurodegenerative apoptosis: an opportunity for therapy?

Apoptotic cell death has been shown to constitute the terminal process in some neurodegenerative diseases, notably Alzheimer's disease and Parkinson's disease (PD). A decrease in mitochondrial membrane potential (delta psiM) causing opening of the permeability transition pore (PTP) in mitochondrial membranes has been implicated as a critical effector of apoptosis in a variety of non-neural cells. Opening of the PTP leads to the release of so-called apoptosis initiation factors that induce the degradative events of apoptosis, such as nuclear chromatin condensation and DNA fragmentation. We have extended those findings to a neuronal model of apoptosis caused by trophic withdrawal, by showing that a decrease in delta psiM is an early event occurring 2 to 6 hours before the degradative events of apoptosis. A deficiency in mitochondrial complex I activity has been demonstrated in the substantia nigra of postmortem brains and several peripheral tissues obtained from PD patients. Because delta psiM is generated by the pumping of protons out across the inner mitochondrial membrane at the mitochondrial complexes, particularly complex I, we hypothesized that the decrease in complex activity could result in a decrease in delta psiM that would render PD substantia nigra neurons vulnerable to apoptosis. In preliminary studies, we have found a decrease in delta psiM in fibroblasts obtained from some PD patients. If a decrease in delta psiM consequent on decreased complex activity is an intrinsic defect in some PD patients, it would open a number of new avenues for the reduction of neuronal apoptosis in PD. The oncoprotein BCL-2 and the scavenger protein SOD-1 have been shown to reduce apoptosis by facilitating closure of the PTP. A number of agents have been shown to maintain BCL-2 and/or SOD-1 synthesis in damaged nerve cells and thereby reduce apoptosis. Other agents, such as cyclosporin A and some benzodiazepine receptor-binding agents, have been found to act directly on the PTP to reduce apoptosis. Accordingly, agents that maintain delta psiM and PTP closure may offer new and effective means of treating neurodegenerative apoptosis.

Alzheimer Disease↗