P-glycoprotein misfolds in Escherichia coli: evidence against alternating-topology models of the transport cycle

P-glycoprotein (P-gp) is a drug transporter which pumps toxic hydrophobic compounds out of cells, conferring mutidrug resistance. P-gp is predicted to consist of 12 transmembrane alpha-helices and there is a strong body of experimental support for this model. However, a number of studies, including those on P-gp expressed in E. coli, have reported topologies with fewer than 12 transmembrane alpha-helices, leading to the hypothesis that the transmembrane topology of the protein changes during function. It is well established that P-gp undergoes conformational changes during its transport cycle and it has been recently shown that these changes are large in magnitude and could, potentially, reflect a changing transmembrane topology. One therefore, reassessed the transmembrane topology of P-gp expressed in E. coli and compared it directly with the topology of the protein expressed in mammalian cells. It was clear that the transmembrane topology of the protein was different in the different cell types and that the misfolding of P-gp in E. coli was due to the misrecognition of multiple P-gp sequences as topogenic signals. Thus, the alternative transmembrane topologies reported for P-gp in E. coli are artefacts of the heterologous expression system used, and models based on such data in which the transmembrane topology changes during drug transport are unlikely to be correct. Instead, the large conformational changes observed during the transport cycle are more likely due to changes in alpha-helix packing.     

The glycosylation and orientation in the membrane of the third cytoplasmic loop of human P-glycoprotein is affected by mutations and substrates

Multiple topologies have been detected for the COOH-terminal half of the human multidrug resistance P-glycoprotein (P-gp). In one topology, the predicted third cytoplasmic loop (CL3) is on the cytoplasmic side (P-gp-CL3-cyt) of the membrane. In an alternate topology, CL3 is on the extracellular side of the membrane (P-gp-CL3-ext). It is not known if both forms of P-gp are active because it is difficult to distinguish either topology in the full-length molecule. When the halves of P-gp are expressed as separate polypeptides, the two topologies of the C-Half are readily distinguished on SDS-PAGE, because only the C-Half (CL3-ext) is glycosylated. To test whether both topologies can fold into an active enzyme, we assayed for interaction between the N- and C-Halves of P-gp since functional P-gp requires interaction between both halves. In a mutant P-gp (E875C) that gave about equal amounts of both topologies, only the C-Half (CL3-cyt) could be recovered by nickel chromatography after coexpression with the histidine-tagged N-Half P-gp. The isolated N-Half and E875C C-Half (CL3-cyt) polypeptides, when expressed together, exhibited verapamil- and vinblastine-stimulated ATPase activities that were similar to the wild-type enzyme. We also found that biosynthesis of mutant E875C C-Half in the presence of the N-Half P-gp resulted in enhanced expression of C-Half (CL3-cyt). By contrast, interaction of C-Half (CL3-ext) with N-Half P-gp was not detected. These results show that the topology of the C-Half portion of P-gp greatly influences its interactions with the amino-terminal half of the molecule.     

α-Tocopherol Transfer Protein Expression in Rat Liver Exposed to Hyperoxia

&#102 -Tochopherol transfer protein ( &#102 TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for &#102 -tochopherol. The factors that regulate the expression of hepatic &#102 TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (>95% O 2 for 48 h) could alter the expression of hepatic &#102 TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic &#102 TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of &#102 TTP messenger RNA (mRNA) after hyperoxia, although the &#102 TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic &#102 TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.     

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly⁻) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly⁻ P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly⁻ P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly⁻ D (91, 94, 99N→D) and Gly⁻Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly⁻ D and Gly⁻Δ Pgps were also expressed to the same level as the Gly⁻ mutant protein. ³⁵S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of ³⁵S-methionine/cysteine in full length Gly⁻ P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of ³⁵S-methionine/cysteine in Gly⁻ P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies. Received: 28 July 1999/Revised: 20 October 1999     

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.     

Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition

Once inserted, transmembrane segments of polytopic membrane proteins are generally considered stably oriented due to the large free energy barrier to topological reorientation of adjacent extramembrane domains. However, the topology and function of the polytopic membrane protein lactose permease of Escherichia coli are dependent on the membrane phospholipid composition, revealing topological dynamics of transmembrane domains after stable membrane insertion (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). In this study, we show that the high affinity phenylalanine permease PheP shares many similarities with lactose permease. PheP assembled in a mutant of E. coli lacking phosphatidylethanolamine (PE) exhibited significantly reduced active transport function and a complete inversion in topological orientation of the N terminus and adjoining transmembrane hairpin loop compared with PheP in a PE-containing strain. Introduction of PE following the assembly of PheP triggered a reorientation of the N terminus and adjacent hairpin to their native orientation associated with regain of wild-type transport function. The reversible orientation of these secondary transport proteins in response to a change in phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly.     

Expression,purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H + -ATPase subunit F (Vma7p)

The vacuolar H + -ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F 1 F 0 -ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13 460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% &#102 -helix and 32-42% &#103 -sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five &#102 - &#103 sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.     

Mutations in Intracellular Loops 1 and 3 Lead to Misfolding of Human P-glycoprotein (ABCB1) That Can Be Rescued by Cyclosporine A,Which Reduces Its Association with Chaperone Hsp70

P-glycoprotein (P-gp) is an ATP binding cassette transporter that effluxes a variety of structurally diverse compounds including anticancer drugs. Computational models of human P-gp in the apo- and nucleotide-bound conformation show that the adenine group of ATP forms hydrogen bonds with the conserved Asp-164 and Asp-805 in intracellular loops 1 and 3, respectively, which are located at the interface between the nucleotide binding domains and transmembrane domains. We investigated the role of Asp-164 and Asp-805 residues by substituting them with cysteine in a cysteine-less background. It was observed that the D164C/D805C mutant, when expressed in HeLa cells, led to misprocessing of P-gp, which thus failed to transport the drug substrates. The misfolded protein could be rescued to the cell surface by growing the cells at a lower temperature (27 °C) or by treatment with substrates (cyclosporine A, FK506), modulators (tariquidar), or small corrector molecules. We also show that short term (4–6 h) treatment with 15 μm cyclosporine A or FK506 rescues the pre-formed immature protein trapped in the endoplasmic reticulum in an immunophilin-independent pathway. The intracellularly trapped misprocessed protein associates more with chaperone Hsp70, and the treatment with cyclosporine A reduces the association of mutant P-gp, thus allowing it to be trafficked to the cell surface. The function of rescued cell surface mutant P-gp is similar to that of wild-type protein. These data demonstrate that the Asp-164 and Asp-805 residues are not important for ATP binding, as proposed earlier, but are critical for proper folding and maturation of a functional transporter.     

In Vitro Antioxidant Activity of 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), a Vitamin E Metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman ( &#102 -CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of &#102 -CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS &#148 + ) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, &#102 -tocopherol, ascorbic acid, and ( &#109 )-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, &#102 -CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that &#102 -CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

Consensus predictions of membrane protein topology

We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology. Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E. coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority-vote approach.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid

It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.     

Recent Progress in Understanding the Mechanism of P-Glycoprotein-mediated Drug Efflux

P-glycoprotein (P-gp) is an ATP-dependent drug pump that can transport a broad range of hydrophobic compounds out of the cell. The protein is clinically important because of its contribution to the phenomenon of multidrug resistance during AIDS/HIV and cancer chemotherapy. P-gp is a member of the ATP-binding cassette (ABC) family of proteins. It is a single polypeptide that contains two repeats joined by a linker region. Each repeat has a transmembrane domain consisting of six transmembrane segments followed by a hydrophilic domain containing the nucleotide-binding domain. In this mini-review, we discuss recent progress in determining the structure and mechanism of human P-glycoprotein.     

Do drug substrates enter the common drug-binding pocket of P-glycoprotein through "gates"?

Overexpression of P-glycoprotein (P-gp; ABCB1) can cause multidrug resistance during cancer and AIDS chemotherapy because of its ability to transport a broad range of structurally unrelated compounds from the cell. P-gp is a member of the ABC family of proteins. It is a single polypeptide containing four domains—two transmembrane (TM) domains each of which contains six TM segments and two nucleotide-binding domains. Chemical modification and cross-linking studies of cysteine mutants of P-gp indicate that the common drug-binding pocket is at the interface between the TM domains. It has been postulated that drug substrates enter the lipid bilayer, are extracted by P-gp and transported to the extracellular medium. It is not clear how drug substrates enter the drug-binding pocket. Here, we propose that drug-substrates diffuse from the lipid bilayer into the drug-binding pocket through “gates” formed by TM segments at either end of the drug-binding pocket.     

Membrane topology mapping of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis

The membrane topologies of the six subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na⁺-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.     

Effect of Chronic Ethanol Feeding on Oxysterols in Rat Liver

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 &#102 - and 7 &#103 -hydroperoxycholest-5-en-3 &#103 -ol (7 &#102 -OOH and 7 &#103 -OOH) as well as 7 &#102 - and 7 &#103 -hydroxycholesterol (7 &#102 -OH and 7 &#103 -OH), and 3 &#103 -hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 &#102 -OOH (+169%, P =0.005 ) and 7 &#103 -OOH (+199%, P =0.011 ). Increases in the concentrations of hepatic 7-keto (+74%, P =0.01 ) and decreases in cholesterol ( &#109 43%; P =0.03 ) also occurred. In contrast, the concentrations of both 7 &#102 -OH and 7 &#103 -OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol ( P =0.0006), 7 &#102 -OH/cholesterol ( P =0.0018) and 7 &#103 -OH/cholesterol ( P =0.0047). In conclusion, this is the first report of increased 7 &#102 -OOH, 7 &#103 -OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.     

以重组P-gp为抗原建立检测MDR 1抗体间接ELISA方法的研究

目的:构建MDR 1基因原核表达质粒,表达P-gp重组蛋白,建立检测MDR1抗体的间接ELISA方法。方法:利用重组PCR技术扩增MDR 1基因的1kb片段,克隆至pET-28b（＋）中,构建原核表达质粒pETP-gp,转染感受态菌BL21（DE3）和BL21（DE3）plyss;以E.coli高效表达的P-gp基因主要抗原编码区重组蛋白为抗原,以HRP标记的兔抗人IgG为二抗,建立间接ELISA检测方法。结果:正确构建了pETP-gp原核表达质粒,并可在E.coli中高效表达,表达蛋白可用作检测MDR 1抗体ELISA抗原。结论:成功表达出重组蛋白P-gp,建立了检测MDR 1抗体的间接ELISA方法。     

Rescue of Folding Defects in ABC Transporters Using Pharmacological Chaperones

The ATP-binding cassette (ABC) family of membrane transport proteins is the largest class of transporters in humans (48 members). The majority of ABC transporters function at the cell surface. Therefore, defective folding and trafficking of the protein to the cell surface can lead to serious health problems. The classic example is cystic fibrosis (CF). In most CF patients, there is a deletion of Phe508 in the CFTR protein (ΔF508 CFTR) that results in defective folding and intracellular retention of the protein (processing mutant). A potential treatment for most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect. The feasibility of such an approach was first demonstrated with the multidrug transporter P-glycoprotein (P-gp), an ABC transporter, and a sister protein of CFTR. It was found that P-gps with mutations at sites equivalent to those found in CFTR processing mutants were rescued when they were expressed in the presence of drug substrates or modulators of P-gp. These compounds acted as pharmacological chaperones and functioned by promoting interactions among the various domains in the protein during the folding process. Several groups have attempted to identify compounds that could rescue the folding defect in ΔF508 CFTR. The best compound identified through high-throughout screening is a quinazoline derivative (CFcor-325). Expression of ΔF508 CFTR as well as other CFTR processing mutants in the presence of 1 μM CFcor-325 promoted folding and trafficking of the mutant proteins to the cell surface in an active conformation. Therefore, CFcor-325 and other quinazoline derivates could be important therapeutic compounds for the treatment of CF.     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles - mainly water - of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in &#102 M1, &#102 M4, &#110 M1 and &#110 M4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear &#102 -helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr 422, a residue close to the extracellular-facing membrane hemilayer in &#102 M4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.     

Characterization and topology of the membrane domain Nqo10 subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of 14 different subunits (Nqo1-Nqo14). Of these, seven subunits (Nqo7, Nqo8, and Nqo10-14) which are equivalent to the mitochondrial DNA-encoded subunits of complex I constitute the membrane segment of the enzyme complex; the remaining subunits make up the peripheral part of the enzyme. We report here on the biochemical characterization and heterologus expression of the Nqo10 subunit. The Nqo10 subunit could not be extracted from the Paracoccus membranes by NaI or alkaline treatment, which is consistent with the presumed membrane localization. By using the maltose-binding protein (MBP) fusion system, the Nqo10 subunit was overexpressed in Escherichia coli. The MBP-fused Nqo10 was expressed in membrane fractions of the host cell and was extractable by Triton X-100. The extracted fusion protein was then isolated by one-step affinity purification through an amylose column. By using immunochemical methods in conjunction with cysteine-scanning mutagenesis and chemical modification techniques, the topology of the Nqo10 subunit expressed in E. coli membranes was determined. The data indicate that the Nqo10 subunit consists of five transmembrane segments with the N- and C-terminal regions facing the periplasmic and cytoplasmic sides of the membrane, respectively. In addition, the data also suggest that the proposed topology of the MBP-fused Nqo10 subunit expressed in E. coli membranes is consistent with that of the Nqo10 subunit in the native Paracoccus membranes. From the experimentally determined topology together with computer prediction programs, a topological model for the Nqo10 subunit is proposed.