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Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant and of its parent strain. The addition of the detergent-solubilized material resulted in a strong increase in the membrane conductance which was not observed if only the detergent was added to the aqueous phase. Surprisingly, the membrane conductance induced by the detergent extracts of the mutant membrane was only a factor of 20 less than that caused by the outer membrane of the parent strain under otherwise identical conditions. Single-channel recordings of lipid bilayer membranes in the presence of mitochondrial outer membranes of the yeast mutant suggested the presence of a transient pore. The reconstituted pores had a single-channel conductance of 0.21 nS in 0.1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. The pores present in the mitochondrial outer membranes of the yeast mutant shared some similarities with the pores formed by mitochondrial and bacterial porins although their effective diameter is much smaller than those of the 'normal' mitochondrial porins which have a single-channel conductance of about 0.4 nS in 0.1 M KCl, corresponding to an effective diameter of 1.7 nm. Zero-current membrane-potential measurements suggested that the second mitochondrial porin is slightly cation-selective. Its possible role in the metabolism of mitochondria is discussed. 相似文献
3.
Ogishima T Kinoshita JY Mitani F Suematsu M Ito A 《The Journal of biological chemistry》2003,278(23):21204-21211
Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017alpha, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017alpha-catalyzed reactions, 17alpha-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17alpha-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17alpha-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells. 相似文献
4.
The role of hexokinase as a possible modulator of Ca2+ movements in isolated rat brain mitochondria 总被引:1,自引:0,他引:1
The present study shows that in brain mitochondria the active calcium uptake and the sodium-dependent calcium efflux are modulated by the porin-bound enzyme hexokinase. The release of the enzyme, promoted by glucose-6-phosphate (G-6-P), under conditions which do not affect mitochondrial functions, is accompanied by a decrease of the rates of fluxes of the cation. This phenomenon is discussed and correlated with the formation of microcompartments between the inner and outer mitochondrial membranes, where the hexokinase-porin complex may constitute a regulating gate system for calcium. 相似文献
5.
In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase
of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved
in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only
17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with
the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physological importance of these
effects is discussed.
Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 395–407. 相似文献
6.
The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria 总被引:20,自引:0,他引:20
Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane. 相似文献
7.
NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed. 相似文献
8.
Gellerich Frank Norbert Kapischke Matthias Kunz Wolfram Neumann Wolfram Kuznetsov Andrey Brdiczka Dieter Nicolay Klaas 《Molecular and cellular biochemistry》1994,133(1):85-104
Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed. 相似文献
9.
Summary Outer mitochondrial membranes isolated by the swelling-shrinkage sonication procedure of Sottocasaet al. [19, 20] forms small sealed vesicles. If cytochromec is present during the procedure it is trapped inside these vesicles and can not be washed out nor is accessible to external enzymes, e.g., cytochrome oxidase (EC 1.9.3.1) or succinate-cytochromec reductase present as contamination by the inner membrane, but is fully accessible to rotenone-insensitive NADH-cytochromec reductase of the outer membrane. This indicates the impermeability of the outer mitochondrial membrane to cytochromec.A modification of the original procedure for the separation of the outer mitochondrial membrane is described. 相似文献
10.
The process of mitochondrial protein import has been studied for many years. Despite this attention, many processes associated with mitochondrial biogenesis are poorly understood. Insight into one of these processes, assembly of beta-barrel proteins into the mitochondrial outer membrane, will be discussed. This review focuses on recent data that suggest that assembly of beta-barrel proteins into the outer mitochondrial membrane is dependent on a newly identified protein complex termed the sorting and assembly machinery (SAM complex). Members of the SAM complex have been identified in both eukaryotic and prokaryotic organisms, suggesting that the process of beta-barrel assembly into membranes has been conserved through evolution. 相似文献
11.
Duncan O Taylor NL Carrie C Eubel H Kubiszewski-Jakubiak S Zhang B Narsai R Millar AH Whelan J 《Plant physiology》2011,157(3):1093-1113
The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. 相似文献
12.
Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae 总被引:4,自引:0,他引:4
The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties. 相似文献
13.
Mitochondria-cytoskeleton interactions in the sperm midpiece 总被引:2,自引:0,他引:2
14.
In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5). 相似文献
15.
Roland Benz Angela Schmid Melitta Dihanich 《Journal of bioenergetics and biomembranes》1989,21(4):439-450
Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed. 相似文献
16.
Meineke B Engl G Kemper C Vasiljev-Neumeyer A Paulitschke H Rapaport D 《FEBS letters》2008,582(6):855-860
The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle. 相似文献
17.
How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane?
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Rapaport D 《The Journal of cell biology》2005,171(3):419-423
A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed beta-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted beta-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit beta-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 beta-barrel structure. 相似文献
18.
D.E. Bignell 《Tissue & cell》1980,12(1):153-164
The colonic epithelium has been examined for ultrastructural evidence of physiological activity. The cells show extensive folding of the apical plasma membrane, associated with mitochondria and an internal coating of particles about 120 Å diameter. Anteriorly many apical infoldings are dilated at the tip to form substantial extracellular spaces up to 0.8 μm wide. Narrow intercellular channels are present, opening to the haemolymph side of the epithelium. Pinocytosis is seen frequently at the basal surface. The surface densities of apical plasma membranes were not significantly different in the posterior mid-gut and colon. Similarly the volume densities of mitochondria were equal in the colonic epithelium and rectal pads, but the surface density of outer mitochondrial membranes was greater in the colon. It is suggested that the colon may absorb organic solutes from the gut lumen. 相似文献
19.
Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells 总被引:25,自引:0,他引:25
Antonsson B Montessuit S Sanchez B Martinou JC 《The Journal of biological chemistry》2001,276(15):11615-11623
Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochrome c-conducting channel in the outer mitochondrial membrane. 相似文献
20.
昆虫线粒体发生的生化和亚显微结构的研究 总被引:1,自引:3,他引:1
线粒体在细胞中的发生目前有各种观点的争论,其理论意义涉及到真核细胞的起源和进化、染色体和线粒体两个遗传体系之间的相互关系以及生物膜合成和组装机理等。我们对处于分化中的昆虫胸肌线粒体的观察结果是:(1)对粘虫变态期的呼吸和细胞色素氧化酶活力测定表明蛹期第8天的组织形成阶段是胸肌细胞分化和其线粒体发生的开始。电镜观察表明,线粒体形成分两个阶段:由颗粒结构(可能是酶蛋白与脂的复合体)装配成膜片和膜泡;由膜泡分化出内嵴,进而发育为线粒体。(2)QO2值,P/O比和ATP酶活力的出现与膜结构的分化发育相平行。α-甘油磷酸氧化酶系统比谷氨酸氧化酶系统装配早;电子传递酶系比磷酸化酶系装配早。(3)蝗虫胸肌分化过程的电镜观察证明;先形成内膜小泡(直径约0.1微米左右),后形成外膜,组成简单线粒体;后者进一步分化发育为成熟线粒体。(4)QO2值,P/O比和ATP酶活力与膜结构分化发育相平行。ATP酶的出现与能量转涣功能呈平行关系。膜形成早期和“幼稚”线粒体阶段,ATP酶尚未装配。(5)综合上述结果:线粒体膜由非膜结构逐步组装形成,线粒体内膜的各酶系组装次序不同步,线粒体DNA控制合成的膜蛋白在膜结构形成中似乎起核心和骨架作用;线粒体总组装过程在不同细胞中表现为多种途径和方式。 相似文献