Fenpyroximate binds to the interface between PSST and 49 kDa subunits in mitochondrial NADH-ubiquinone oxidoreductase

Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.     

The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I

Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3)H](trifluoromethyl)phenyldiazirinylfenpyroximate ([(3)H]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3)H]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [(3)H]TDF-labeled SMP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at approximately 50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na(+)/H(+) antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K(3)Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H(+)(Na(+)) translocation in coupling site 1.     

The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I

The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[125I](trifluoromethyl)phenyldiazirinylacetogenin, [125I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [125I]TDA, radioactivity was predominantly incorporated into an approximately 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [125I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at approximately 30 kDa. Immnoprecipitation of the [125I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [125I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Deltalac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Deltalac-acetogenins differs from that of traditional inhibitors.     

Bis-THF motif of acetogenin binds to the third matrix-side loop of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreductase

Natural acetogenins are among the most potent inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I). Our photoaffinity labeling study suggested that the hydroxylated bis-THF ring moiety of acetogenins binds at "site A" in the third matrix-side loop connecting the fifth and sixth transmembrane helices in the ND1 subunit [Kakutani et al. (2010) Biochemistry 49, 4794-4803]. Nevertheless, since this proposition was led using a photoreactive Δlac-acetogenin derivative, it needs to be directly verified using a natural acetogenin-type probe. We therefore conducted photoaffinity labeling using a photoreactive natural acetogenin mimic ([(125)I]diazinylated natural acetogenin, [(125)I]DANA), which has a small photolabile diazirine group, in place of a hydroxy group, attached to the bis-THF ring moiety. Analysis of the photocross-linked protein in bovine heart submitochondrial particles unambiguously revealed that [(125)I]DANA binds to the membrane subunit ND1 with high specificity. The photocross-linking was completely blocked in the presence of just a 5-fold excess of bullatacin, indicating that [(125)I]DANA is an excellent mimic of natural acetogenins and hence binds to the site that accommodates natural products. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by different proteases and their combinations indicated that the cross-linked residue is predominantly located at the supposed site A in the third matrix-side loop.     

The phosphorylation of subunits of complex I from bovine heart mitochondria

In bovine heart mitochondria and in submitochondrial particles, membrane-associated proteins with apparent molecular masses of 18 and 10 kDa become strongly radiolabeled by [(32)P]ATP in a cAMP-dependent manner. The 18-kDa phosphorylated protein is subunit ESSS from complex I and not as previously reported the 18 k subunit (with the N-terminal sequence AQDQ). The phosphorylated residue in subunit ESSS is serine 20. In the 10 kDa band, the complex I subunit MWFE was phosphorylated on serine 55. In the presence of protein kinase A and cAMP, the same subunits of purified complex I were phosphorylated by [(32)P]ATP at the same sites. Subunits ESSS and MWFE both contribute to the membrane arm of complex I. Each has a single hydrophobic region probably folded into a membrane spanning alpha-helix. It is likely that the phosphorylation site of subunit ESSS lies in the mitochondrial matrix and that the site in subunit MWFE is in the intermembrane space. Subunit ESSS has no known role, but subunit MWFE is required for assembly into complex I of seven hydrophobic subunits encoded in the mitochondrial genome. The possible effects of phosphorylation of these subunits on the activity and/or the assembly of complex I remain to be explored.     

Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I

¹²⁵I-labeled (trifluoromethyl)phenyldiazirinyl acetogenin, [¹²⁵I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) with high specificity [M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409-6416.]. To identify the binding site of [¹²⁵I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [¹²⁵I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144-Glu192) of the subunit. It is worth noting that an excess amount of short-chain ubiquinones such as ubiquinone-2 (Q₂) and 2-azido-Q₂ suppressed the cross-linking by [¹²⁵I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.     

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and characterization of new piperazine-type inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I)

The mode of action of Deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai, M., et al. (2007) Biochemistry 46 , 6409-6416]. As further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex I, we drastically modified the structure of Deltalac-acetogenins and characterized their inhibitory action. In particular, on the basis of structural similarity between the bis-THF and the piperazine rings, we here synthesized a series of piperazine derivatives. Some of the derivatives exhibited very potent inhibition at nanomolar levels. The hydrophobicity of the side chains and their balance were important structural factors for the inhibition, as is the case for the original Deltalac-acetogenins. However, unlike in the case of the original Deltalac-acetogenins, (i) the presence of two hydroxy groups is not crucial for the activity, (ii) the level of superoxide production induced by the piperazines is relatively high, (iii) the inhibitory potency for the reverse electron transfer is remarkably weaker than that for the forward event, and (iv) the piperazines efficiently suppressed the specific binding of a photoaffinity probe of natural-type acetogenins ([ (125)I]TDA) to the ND1 subunit. We therefore conclude that the action mechanism of the piperazine series differs from that of the original Deltalac-acetogenins. The photoaffinity labeling study using a newly synthesized photoreactive piperazine ([ (125)I]AFP) revealed that this compound binds to the 49 kDa subunit and an unidentified subunit, not ND1, with a frequency of approximately 1:3. A variety of traditional complex I inhibitors as well as Deltalac-acetogenins suppressed the specific binding of [ (125)I]AFP to the subunits. The apparent competitive behavior of inhibitors that seem to bind to different sites may be due to structural changes at the binding site, rather than occupying the same site. The meaning of the occurrence of diverse inhibitors exhibiting different mechanisms of action is discussed in light of the functionality of the membrane arm of complex I.     

NADH: ubiquinone oxidoreductase from bovine heart mitochondria. A fourth nuclear encoded subunit with a homologue encoded in chloroplast genomes.

The amino acid sequence has been determined of the precursor of a nuclear encoded 20 kDa subunit of complex I from bovine heart mitochondria. The sequence of the mature protein is related to a protein of uncertain function, hitherto known as psbG, encoded in the chloroplast genomes of higher plants. Open reading frames encoding homologues of psbG have also been detected in bacteria and in the mitochondrial genome of Paramecium tetraurelia. The chloroplast psbG gene is found between ndhC and ndhJ, which encode homologues of ND3, a hydrophobic subunit of complex I encoded in the bovine mitochondrial genome, and of the nuclear encoded 30 kDa subunit of complex I. This 20 kDa protein is the eleventh out of the forty or more subunits of bovine complex I with a chloroplast encoded homologue, and its sequence provides further support for the presence in chloroplasts of a multisubunit enzyme related to complex I that could be involved in chlororespiration. The strict conservation of three cysteines suggests that the subunit might be an iron-sulphur protein.     

Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide

Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.     

Changes in conformation upon agonist binding, and nonequivalent labeling, of the membrane-spanning regions of the nicotinic acetylcholine receptor subunits

All four subunits of the acetylcholine receptor (AChR) are labeled by the lipid-soluble photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) with different stoichiometries and levels of saturable modification sites, dependent on the conformational state of the AChR. This probe is specific for hydrophobic targets such as the membrane-spanning regions of intrinsic proteins. In the resting state, the gamma subunit is labeled 4.5 times greater and the beta and delta subunits 1.65-1.69 greater than the alpha subunit. Carbamylcholine-induced desensitization of the AChR lowers the level and alters the stoichiometry of [125I]TID incorporation into each subunit. This effect is shown to be specific in two ways. First, it is eliminated by prior equilibration with excess alpha-bungarotoxin, which does not change the [125I]TID-labeling pattern of the AChR from that of the resting state. Second, bacteriorhodopsin is labeled by [125I]TID to the same extent both in the presence and absence of carbamylcholine. The noncompetitive blocker phencyclidine does not alter [125I]TID labeling of the AChR relative to the resting state. The 43-kDa protein, which is believed to cross-link the AChR to the cytoskeleton at the synapse, is not modified by [125I]TID, in agreement with earlier conclusions that the 43-kDa protein is not an intrinsic membrane protein.     

Resolution of the membrane domain of bovine complex I into subcomplexes: implications for the structural organization of the enzyme

Complex I (NADH:ubiquinone oxidoreductase) purified from bovine heart mitochondria was treated with the detergent N, N-dimethyldodecylamine N-oxide (LDAO). The enzyme dissociated into two known subcomplexes, Ialpha and Ibeta, containing mostly hydrophilic and hydrophobic subunits, and a previously undetected fragment referred to as Igamma. Subcomplex Igamma contains the hydrophobic subunits ND1, ND2, ND3, and ND4L which are encoded in the mitochondrial genome, and the nuclear-encoded subunit KFYI. During size-exclusion chromatography in the presence of LDAO, subcomplex Ialpha lost several subunits and formed another characterized subcomplex known as Ilambda. Similarly, subcomplex Ibeta dissociated into two smaller subcomplexes, one of which contains the hydrophobic subunits ND4 and ND5; subcomplex Igamma released a fragment containing ND1 and ND2. These results suggest that in the intact complex subunits ND1 and ND2 are likely to be in a different region of the membrane domain than subunits ND4 and ND5. The compositions of the various subcomplexes and fragments of complex I provide an organization of the subunits of the enzyme in the framework of the known low resolution structure of the enzyme.     

Docking of linear peptide antagonists into the human V(1a) vasopressin receptor. Identification of binding domains by photoaffinity labeling.

A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.     

A homologue of a nuclear-coded iron-sulfur protein subunit of bovine mitochondrial complex I is encoded in chloroplast genomes

The chloroplast genomes of Marchantia polymorpha, Nicotiana tabacum, and Oryza sativa contain open reading frames (ORFs or potential genes) encoding homologues of some of the subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I). Seven of these subunits (ND1-ND4, ND4L, ND5, and ND6) are products of the mitochondrial genome, and two others (the 49- and 30-kDa components of the iron-sulfur protein fraction) are nuclear gene products. These findings have been taken to indicate the presence in chloroplasts of an enzyme related to complex I, possibly an NAD(P)H:plastoquinone oxidoreductase, participating in chlororespiration. This view is reinforced by the present work in which we have shown that chloroplast genomes encode a homologue of the 23-kDa subunit, another nuclear-encoded component of bovine complex I. The 23-kDa subunit is in the hydrophobic protein fraction of the enzyme, the residuum after removal of the flavoprotein and iron-sulfur protein fractions. The sequence motif CysXXCysXXCysXXXCysPro, which provides ligands for tetranuclear iron-sulfur centers in ferredoxins, occurs twice in its polypeptide chain and is evidence of two associated 4Fe-4S clusters. This is the only iron-sulfur protein identified so far in the hydrophobic protein fraction of complex I, and so it is possible that one of these centers is that known as N-2, the donor of electrons to ubiquinone. The sequence of the 23-kDa subunit is closely related to potential proteins, which also contain the cysteine-rich sequence motifs, encoded in the frxB ORFs in chloroplast genomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identifying the lipid-protein interface and transmembrane structural transitions of the Torpedo Na,K-ATPase using hydrophobic photoreactive probes

To identify regions of the Torpedo Na,K-ATPase alpha-subunit that interact with membrane lipid and to characterize conformationally dependent structural changes in the transmembrane domain, we have proteolytically mapped the sites of photoincorporation of the hydrophobic compounds 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) and the phosphatidylcholine analogue [(125)I]TIDPC/16. The principal sites of [(125)I]TIDPC/16 labeling were identified by amino-terminal sequence analysis of proteolytic fragments of the Na,K-ATPase alpha-subunit and are localized to hydrophobic segments M1, M3, M9, and M10. These membrane-spanning segments have the greatest levels of exposure to the lipid bilayer and constitute the bulk of the lipid-protein interface of the Na,K-ATPase alpha-subunit. The extent of [(125)I]TID and [(125)I]TIDPC/16 photoincorporation into these transmembrane segments was the same in the E(1) and E(2) conformations, indicating that lipid-exposed segments located at the periphery of the transmembrane complex do not undergo large-scale movements during the cation transport cycle. In contrast, for [(125)I]TID but not for [(125)I]TIDPC/16, there was enhanced photoincorporation in the E(2) conformation, and this component of labeling mapped to transmembrane segments M5 and M6. Conformationally sensitive [(125)I]TID photoincorporation into the M5 and M6 segments does not reflect a change in the levels of exposure of these segments to the lipid bilayer as evidenced by the lack of [(125)I]TIDPC/16 labeling of these two segments in either conformation. These results suggest that [(125)I]TID promises to be a useful tool for structural characterization of the cation translocation pathway and for conformationally dependent changes in the pathway. A model of the spatial organization of the transmembrane segments of the Na,K-ATPase alpha- and beta-subunits is presented.     

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.     

Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.     

Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor.

To identify regions of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used 1-azidopyrene (1-AP) as a fluorescent, photoactivatable hydrophobic probe. For AchR-rich membranes equilibrated with 1-AP, irradiation at 365 nm resulted in covalent incorporation in all four AchR subunits with each of the subunits incorporating approximately equal amounts of label. To identify the regions of the AchR subunits that incorporated 1-AP, subunits were digested with Staphylococcus aureus V8 protease and trypsin, and the resulting fragments were separated by SDS-PAGE followed by reverse-phase high-performance liquid chromatography. N-terminal sequence analysis identified the hydrophobic segments M1, M3, and M4 within each subunit as containing the sites of labeling. The labeling pattern of 1-AP in the alpha-subunit was compared with that of another hydrophobic photoactivatable probe, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID). The nonspecific component of [125I]TID labeling [White, B., Howard, S., Cohen, S. G., & Cohen, J.B. (1991) J. Biol. Chem. 266, 21595-21607] was restricted to the same regions as those labeled by 1-AP. The [125I]TID residues labeled in the hydrophobic segment M4 were identified as Cys-412, Met-415, Cys-418, Thr-422, and Val-425. The periodicity and distribution of labeled residues establish that the M4 region is alpha-helical in nature and indicate that M4 presents a broad face to membrane lipid.     

Impact of mutations affecting ND mitochondria-encoded subunits on the activity and assembly of complex I in Chlamydomonas. Implication for the structural organization of the enzyme

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a 1T deletion in the 3' UTR of nd5 whereas dum17 is a 1T deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160-210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)