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Genomic organization and tissue expression of the mouse proteasome gene Lmp-7.

LMP7 is one of the two proteasome subunits encoded in the major histocompatibility complex and is speculated to play a role in the generation of endogenous peptides for presentation by class I molecules to cytotoxic T cells. Here we report the genomic organization of the mouse Lmp-7 gene and the tissue distribution of its messenger RNA. In contrast to human LMP7 which is composed of seven exons and six introns, the mouse Lmp-7 gene is organized in six exons and five introns. Interestingly, the region corresponding to the first exon of human LMP7 is highly modified by numerous insertions and deletions and contains two in frame stop codons. Consequently, the mouse Lmp-7 gene does not allow the alternative exon usage described in humans and most likely encodes for only one LMP7 protein. Thus, the Tap-1 3' end gene region and the Lmp-7 initial translation codon are separated by an 1182 nucleotide region which contains a TATA-box, a cAMP regulatory element, two SP1 sites, and two G-C-rich regions. Expression of the Lmp-7 messenger RNA was analyzed on different tissues from unstimulated mice. Lmp-7 messenger RNA is expressed in spleen, thymus, lung, liver, heart, and, at a very low level, in kidney but not in brain and testis. The possible role of Lmp genes in antigen processing is discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Orientation of complex III in the yeast mitochondrial membrane: labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate.

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [125I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochrome b revealed that the two core proteins and cytochrome b were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochrome c1 and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromes b and c1 was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15-30% of ;the total dithionite-reducible b and 100% of the cytochrome c1: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochrome b but insignificant amounts of cytochrome c1. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.

Diazonium Compounds↗

Studies on beef heart ubiquinol-cytochrome c reductase. Topological studies on the core proteins using proteolytic digestion and immunoreplication.

The topology of beef heart Complex III has been studied by tryptic and chymotryptic digestion of isolated Complex III, Mg2+-ATP submitochondrial particles, and mitoplasts. Degradation products were detected by the immunoreplication technique using specific antibodies against core protein 1 (50 K) and core protein 2 (47 K). It can be shown that both peptides are digested from the matrix side of the inner membrane. However, no evidence was found that these peptides were digested by trypsin or chymotrypsin from the cytoplasmic side. It is concluded that the beef heart core proteins are membrane-bound peptides containing tryptic and chymotryptic digestion sites only on the matrix surface of the inner membrane. The data also suggest that beef heart core protein 2 contains multiple domains which are inserted into the membrane from the matrix surface. Proteolytic treatment of submitochondrial particles under conditions which digested at least 50% of the core proteins from the matrix surface did not, however, influence NADH oxidation rates or the respiratory control ratios.

Animals↗

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrial bc1 complex.

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrial bc1 complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in the bc1 complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochrome c reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of the bc1 complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochrome c reductase activity. However, a clear stimulation of the rate of cytochrome b reduction in parallel to an inhibition of cytochrome b oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on the bc1 complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.

Animals↗

Further studies on the binding of DCCD to cytochrome B and subunit VIII of complex III isolated from beef heart mitochondria.

Complex III (the cytochrome b-c1 complex) from beef heart mitochondria was incubated with [14C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochrome b) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [14C]DCCD resulted in a 50% decrease in the binding of DCCD to both cytochrome b and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochrome b and subunit VIII of complex III from beef heart mitochondria and suggest that cytochrome b may play a role in proton translocation.

Animals↗

The inhibition site of MPP+, the neurotoxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding site of NADH dehydrogenase.

The inhibition of NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) leading to ATP depletion has been proposed to explain cell death in the expression of the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Electron paramagnetic resonance studies show no effect of MPP+ on the reduction of the iron-sulfur clusters of NADH dehydrogenase. Mitochondria inhibited by MPP+ were sonicated and both the NADH oxidase and the NADH-Q reductase activities were measured. NADH oxidase activity was not fully restored to control levels, but NADH-Q reductase activity was the same as that of the control. Neither succinate-oxidase nor succinate-Q reductase activities were inhibited. These data indicate that MPP+ interaction with NADH dehydrogenase interferes with the passage of electrons from the iron-sulfur cluster of highest potential to endogenous Q10 but that the inhibition can be relieved by the addition of a small, water-soluble Q analog. Inhibition at this site is sufficient to explain the inhibition of respiration and no inhibition of other mitochondrial functions was observed.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy.

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.

Animals↗

Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components.

1. A protein fraction containing three polypeptides (the major one with Mr < 13 000) was isolated by means of Triton X-100 extraction of submitochondrial particles specifically treated to remove succinate dehydrogenase. 2. The mixing of the protein fraction with the soluble reconstitutively active succinate dehydrogenase results in formation of highly active succinate-DCIP reductase which is sensitive to thenoyltrifluoroacetone or carboxin. 3. The maximal turnover number of succinate dehydrogenase in the succinate-DCIP reductase reaction revealed in the presence of a saturating amount of the protein fraction is slightly higher than that measured with phenazine methosulfate as artificial electron acceptor. 4. The protein fraction greatly increases the stability of soluble succinate dehydrogenase under aerobic conditions. 5. The titration of soluble succinate dehydrogenase by the protein fraction shows that smaller amounts of the protein fraction are required to block the reduction of ferrycyanide by Hipip center than that required to reveal the maximal catalytic capacity of the enzyme. 6. The apparent Km of the reconstituted system for DCIP depends on the amount of protein fraction; the more protein fraction added to the enzyme, the lower the Km value obtained. 7. A comparison of different reconstituted succinate-ubiquinone reductases described in the literature is presented and the possible arrangement of the native and reconstituted succinate-ubiquinone region of the respiratory chain is discussed.

2,6-Dichloroindophenol↗

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties.

1. A mehod for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography. 2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with Mr of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 mumol of cytochrome b, 4 mumol of cytochrome c1, 7-8 mumol of nonhemne iron, corresponding to 3.5-4 mumol of the Rieske iron-sulfur protein, less than 1.0 mumol of ubiquinone and about 60 mumol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2g of Triton X-100 per g protein. 3. Cytochrome b exhibits an alpha-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. -10 and + 100 mV, at pH 7.0. The absorbance maximum of cytochrome c1 lies at 553 nm and its half-reduction potential amounts to +250 mV. 4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20%C. 5. A dissociation constant K's of 5.5 mM has been determined for the Tritonsolubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas. 6. A K'm value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.

Animals↗

Effects of dibromothymoquinone on the structure and function of the mitochondrial bc1 complex.

We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).

Animals↗

Bypasses of the antimycin a block of mitochondrial electron transport in relation to ubisemiquinone function.

Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.

2,6-Dichloroindophenol↗

Antimycin binds to a small subunit of the ubiquinol: cytochrome c oxidoreductase.

Bovine heart ubiquinol: cytochrome c oxidoreductase in Triton X-100 is split with guanidine into a number of fractions. A new method for measuring antimycin binding is developed using extraction with pentanol of the reversibly bound antimycin. By this method and the normal titration method, antimycin-binding capacity is found in a fraction containing a small subunit with a molecular mass of about 12000. This polypeptide was associated with cytochrome c1 but is probably not the 'hinge protein'. Fractions that contain cytochrome b did not show binding by the pentanol-extraction method.

Amino Acids↗

Inhibition of complex I by isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Mitochondrial respiratory failure secondary to complex I inhibition may contribute to the neurodegenerative process underlying nigral cell death in Parkinson's disease (PD). Isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) may be inhibitors of complex I, and have been implicated in the cause of PD as endogenous neurotoxins. To determine the potency and structural requirements of isoquinoline derivatives to inhibit mitochondrial function, we examined the effects of 22 neutral and quaternary compounds from three classes of isoquinoline derivatives (11 isoquinolines, 2 dihydroisoquinolines, and 9 1,2,3,4-tetrahydroisoquinolines) and MPP+ on the enzymes of the respiratory chain in mitochondrial fragments from rat forebrain. With the exception of norsalsolinol and N,n-propylisoquinolinium, all compounds inhibited complex I in a time-independent, but concentration-dependent manner, with IC50s ranging from 0.36-22 mM. Several isoquinoline derivatives were more potent inhibitors of complex I than 1-methyl-4-phenylpyridinium ion (MPP+) (IC50 = 4.1 mM), the most active being N-methyl-6-methoxy-1,2,3,4-tetrahydroisoquinoline (IC50 = 0.36 mM) and 6-methoxy-1,2,3,4-tetrahydroisoquinoline (IC50 = 0.38 mM). 1,2,3,4-Tetrahydroisoquinoline was the least potent complex I inhibitor (IC50 approximately 22 mM). At 10 mM, only isoquinoline (23.1%), 6,7-dimethoxyisoquinoline (89.6%), and N-methylsalsolinol (34.8%) inhibited (P < 0.05) complex II-III, but none of the isoquinoline derivatives inhibited complex IV. There were no clear structure-activity relationships among the three classes of isoquinoline derivatives studied, but lipophilicity appears to be important for complex I inhibition. The effects of isoquinoline derivatives on mitochondrial function are similar to those of MPTP/MPP+, so respiratory inhibition may underlie their reported neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗