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PubMed1982

Relationship between the density distribution of intramembrane particles and electron transfer in the mitochondrial inner membrane as revealed by cholesterol incorporation.

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to-cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron-transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.

Animals↗
PubMed2004

Modelling NADH turnover in plant mitochondria.

NADH is central to the functioning of mitochondrial respiration. It is produced by enzymes in, or associated with, the tricarboxylic acid cycle in the matrix, and it is oxidized by two respiratory chain enzymes in the inner membrane, the rotenone-sensitive complex I and the rotenone-insensitive internal NADH dehydrogenase (ND(in)). A simplified kinetic model for NADH turnover in the matrix of plant mitochondria is presented. Only the two main NADH-producing enzymes, NAD-malate dehydrogenase [EC 1.1.1.37] (MDH) and NAD-malic enzyme [EC 1.1.1.39] (ME), are considered. This model reproduces the complex behaviour of malate oxidation by isolated mitochondria in response to additions of ADP (state 3/state 4), NAD(+) and/or rotenone, as well as to changes in pH. It is found that MDH always operates at or close to equilibrium. Changes in the activity of complex I, ND(in), or ME are predicted to cause clear changes in the pattern of malate oxidation. In general, the model predicts high sensitivity to changes in the ME activity. In contrast, MDH activity can be reduced 100-fold without detectable changes in malate oxidation. It is demonstrated that it is not the high activity, but the equilibrium properties of MDH that are important for the redox-buffering function of MDH in the mitochondrial matrix. Binding of NAD(+) and NADH in the matrix reduces the concentrations of free NAD(+) and NADH, depending on the concentration of binding sites and the binding strength. On the basis of the modelling results it is estimated that a significant proportion of the mitochondrial NAD is bound.

Journal Article↗
PubMed2006

Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism.

The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents, organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating access mechanism and a novel peristaltic mode of drug transport by this type of transporter.

Biological Transport↗
PubMed2005

A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis.

The type III secretion system (T3SS) is an important virulence factor used by several gram-negative bacteria to deliver effector proteins which subvert host cellular processes. Enterohemorrhagic Escherichia coli O157 has a well-defined T3SS involved in attachment and effacement (ETT1) and critical for virulence. A gene cluster potentially encoding an additional T3SS (ETT2), which resembles the SPI-1 system in Salmonella enterica, was found in its genome sequence. The ETT2 gene cluster has since been found in many E. coli strains, but its in vivo role is not known. Many of the ETT2 gene clusters carry mutations and deletions, raising the possibility that they are not functional. Here we show the existence in septicemic E. coli strains of an ETT2 gene cluster, ETT2(sepsis), which, although degenerate, contributes to pathogenesis. ETT2(sepsis) has several premature stop codons and a large (5 kb) deletion, which is conserved in 11 E. coli strains from cases of septicemia and newborn meningitis. A null mutant constructed to remove genes coding for the putative inner membrane ring of the secretion complex exhibited significantly reduced virulence. These results are the first demonstration of the importance of ETT2 for pathogenesis.

Animals↗
PubMed1984

Cholesterol accumulation in adrenocortical mitochondria after ACTH-stimulation.

Rat adrenocortical cell suspensions (10(6) cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min. About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium. Aminoglutethimide at 50 microM inhibited the steroidogenesis to 16%. Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined. The mitochondria of the cells incubated without the drugs contained 25.2 micrograms cholesterol/mg protein. Cholesterol content increased by 10% in the mitochondria of the ACTH-stimulated cells. The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143% of cholesterol compared to those of the nontreated cells. When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200% compared to that of the control rats. These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells. The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy. Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter.

Adrenal Cortex↗
PubMed1981

Oxygen permeability of the shell and membranes of chicken eggs during development.

Oxygen permeability (KO2) was measured through the shell and shell membranes of chicken eggs throughout incubation. Shell KO2 was constant at 1.90 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Outer shell membrane KO2 was constant at 1.78 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Inner membrane KO2 increased from 0.11 x 10(-6) cm3 O2 STP . sec-1 . cm-2. Torr-1 to 1.56 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Calculations of KO2 from oxygen uptake rates and air cell PO2's were in close agreement with direct measurement of KO2. Resistance to oxygen flux was partitioned at each level. The outer membrane added 6% to the resistance of the shell. The inner membrane initially accounted for 88% of the shell/membrane complex resistance, but fell to 12% of the resistance by the end of incubation. The hypothesis is discussed that the increased permeability of the inner membrane is related to the evaporation of water from the membrane surface. The possibility is rejected that the shell membranes may be potential sites for respiratory adaptation to incubation at altitude.

Adaptation, Physiological↗
PubMed2002

Blue Native electrophoresis to study mitochondrial and other protein complexes.

The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be applied to other protein complexes. The use of BN-PAGE has increased exponentially over the past few years and new applications have been developed. Here we review how to set up the basic system and outline modifications that can be applied to address specific research questions. Increasing the upper mass limit of complexes that can be separated by BN-PAGE can be achieved by using agarose instead of acrylamide. BN-PAGE can also be used to study assembly of mitochondrial protein complexes. Other applications include in-gel measurements of enzyme activity by histochemical staining and preparative native electrophoresis to isolate a protein complex. Finally, new ways of identifying protein spots in Blue Native gels using mass spectrometry are briefly discussed.

Biochemistry↗
PubMed2005

Functional analysis of the domains in Cox11.

Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.

Amino Acid Motifs↗
PubMed1999

The TIM17.23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix.

We have analysed the structural organization of the TIM17.23 complex, the preprotein translocase of the mitochondrial inner membrane specific for protein targeting to the matrix. The components Tim17, Tim23 and Tim44 are present in this complex in equimolar amounts. A sub-complex containing Tim23 and Tim44 but no Tim17, or a sub-complex containing Tim23 and Tim17 but no Tim44 was not detected. Tim44 is peripherally associated at the matrix side. Tim44 forms dimers which recruit two molecules of mt-Hsp70 to the sites of protein import. A sequential, hand-over-hand mode of interaction of these two mt-Hsp70.Tim44 complexes with a translocating polypeptide chain is proposed.

Amino Acid Sequence↗
PubMed2001

Biogenesis of the dicarboxylate carrier (DIC): translocation across the mitochondrial outer membrane and subsequent release from the TOM channel are membrane potential-independent.

The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites. The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane. Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV). In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel. Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane. In this study, we characterized the import pathway of the dicarboxylate carrier (DIC). Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*). The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins.

Carrier Proteins↗
PubMedDate not supplied

Mitochondrial dynamics generate equal distribution but patchwork localization of respiratory Complex I.

Highly dynamic mitochondrial morphology is a prerequisite for fusion and fission. Mitochondrial fusion may represent a rescue mechanism for impaired mitochondria by exchanging constituents (proteins, lipids and mitochondrial DNA) and thus maintaining functionality. Here we followed for the first time the dynamics of a protein complex of the respiratory chain during fusion and fission. HeLa cells with differently labelled respiratory Complex I were fused and the dynamics of Complex I were investigated. The mitochondrial proteins spread throughout the whole mitochondrial population within 3 to 6 h after induction of cell fusion. Mitochondria of fused cells displayed a patchy substructure where the differently labelled proteins occupied separate and distinct spaces. This patchy appearance was already--although less pronounced--observed within single mitochondria before fusion, indicating a specific localization of Complex I with restricted diffusion within the inner membrane. These findings substantiate the view of a homogenous mitochondrial population due to constantly rearranging mitochondria, but also indicate the existence of distinct inner mitochondrial sub-compartments for respiratory chain complexes.

Animals↗
PubMed2004

Organization and function of the small Tim complexes acting along the import pathway of metabolite carriers into mammalian mitochondria.

Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria. In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins. Here, we show that the human small proteins form two distinct hetero-oligomeric complexes. A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa. This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12. Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space. Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes. During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner. For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase. However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22. We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes.

Animals↗
PubMed1997

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Amino Acid Sequence↗
PubMedDate not supplied

Mechanism of citrinin-induced dysfunction of mitochondria. III. Effects on renal cortical and liver mitochondrial swelling.

The effects of the mycotoxin citrinin on renal cortical and liver mitochondrial swelling were studied. Citrinin decreases the rate of swelling induced by the valinomycin-K+ complex, suggesting that the mycotoxin interferes with the mitochondrial membrane fluidity. Citrinin promotes reduction of the amplitude of swelling in the presence of Na+ ions. This alteration reflects interference with complex I of the respiratory chain and ATP synthase complex activity without disarranging the inner mitochondrial membrane, in view of the fact that the shrinkage was not affected. The effect increases with citrinin concentration. Renal tissue is more susceptible than hepatic tissue.

Animals↗
PubMed2000

Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.

Around 30-40 years after the first isolation of the five complexes of oxidative phosphorylation from mammalian mitochondria, we present data that fundamentally change the paradigm of how the yeast and mammalian system of oxidative phosphorylation is organized. The complexes are not randomly distributed within the inner mitochondrial membrane, but assemble into supramolecular structures. We show that all cytochrome c oxidase (complex IV) of Saccharomyces cerevisiae is bound to cytochrome c reductase (complex III), which exists in three forms: the free dimer, and two supercomplexes comprising an additional one or two complex IV monomers. The distribution between these forms varies with growth conditions. In mammalian mitochondria, almost all complex I is assembled into supercomplexes comprising complexes I and III and up to four copies of complex IV, which guided us to present a model for a network of respiratory chain complexes: a 'respirasome'. A fraction of total bovine ATP synthase (complex V) was isolated in dimeric form, suggesting that a dimeric state is not limited to S.cerevisiae, but also exists in mammalian mitochondria.

ATP Synthetase Complexes↗
PubMed2001

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors.

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

Fungal Proteins↗
PubMed2006

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I.

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.

Animals↗
PubMed1993

Prediction of transmembrane beta-strands from hydrophobic characteristics of proteins.

The assembly of outer-membrane proteins consisting of beta-strands as transmembrane segments is somewhat more complex when compared to the assembly of inner membrane proteins having alpha-helices as transmembrane parts. This is probably due to the difference in the amino acid sequences of the transmembrane part strands and helices. Because of this feature, most predictive schemes which are successful in predicting transmembrane helical segments fail to predict transmembrane strand segments. Here we propose a new predictive scheme for forecasting the transmembrane strand segments in outer-membrane proteins with the use of the general surrounding hydrophobicity scale developed both for the globular and membrane proteins in the preceding article. Two major features of the scheme are (i) that it does not solely depend on the amphipathic character of a sequence segment while identifying it as a transmembrane strand, and it is capable of predicting strands in varied lengths, a facility to reflect the variation in the membrane surfaces. This scheme predicts the transmembrane beta-strands in porin from R. capsulatus at 76% accuracy (giving due weights to over- and under-predictions when compared to X-ray results). The predicted beta-structure contents in OmpA, porin from E. coli and maltoporin compared with the Raman spectroscopic results at 95% level. These accuracy levels are far superior than the levels obtained from other existing methods. Apart from the above four proteins for which experimental informations are available, ten other outer-membrane proteins, for which there is no information about their secondary structure, are considered in the forecast. The results are analysed and the common features in the folds of the set of fourteen outer-membrane proteins are deduced.

Amino Acid Sequence↗