Impermeant Thiol Reagents Research Articles

Investigation of the structure and function of aShewanella oneidensisarsenical-resistance family transporter

The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly alpha-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that TM VIII faces an aqueous channel.

The contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water-filled channel through which ATP is transported was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli. A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used. Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport. The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity. Five patterns of inhibition of ATP transport by the MTS reagents were observed. (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C. (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents. (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C). (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C. However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate. (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES. Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition. Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.

Cytoplasmic Loop Connecting Helices IV and V of the Melibiose Permease from Escherichia coli Is Involved in the Process of Na+-coupled Sugar Translocation

Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.

Cftr

Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding.

Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in the absence or presence of ligand. In right-side-out membrane vesicles, Cys residues in the cytoplasmic halves of both helices react with NEM in the absence of ligand, while Cys residues in the periplasmic halves do not. Five Cys replacement mutants at the periplasmic end of helix V and one at the cytoplasmic end of helix V label only in the presence of ligand. Interestingly, in addition to native Cys148, a known binding-site residue, labeling of mutant Ala122 --> Cys, which is located in helix IV across from Cys148, is markedly attenuated by ligand. Furthermore, alkylation of the Ala122 --> Cys mutant blocks transport, and protection is afforded by substrate, indicating that Ala122 is also a component of the sugar binding site. Methanethiosulfonate ethylsulfonate, an impermeant thiol reagent shown clearly in this paper to be impermeant in E. coli spheroplasts, was used to identify substituted Cys side chains exposed to water and accessible from the periplasmic side. Most of the Cys mutants in the cytoplasmic halves of helices IV and V, as well as two residues in the intervening loop, are accessible to the aqueous phase from the periplasmic face of the membrane. The findings indicate that the cytoplasmic halves of helices IV and V are more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that exhibit ligand-induced changes are located for the most part in the vicinity of the residues directly involved in substrate binding, as well as the cytoplasmic loop between helices IV and V.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII.

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.

Topology of the Na+/Proline Transporter of Escherichia coli

Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.

The sulfonylurea-inhibited NADH oxidase activity of HeLa cell plasma membranes has properties of a protein disulfide-thiol oxidoreductase with protein disulfide-thiol interchange activity.

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY 181984, with an EC50 of 30 nM, also inhibited a protein disulfide-thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide-thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide-thiol interchange coupled to cell enlargement.

The Hormone-responsive NADH Oxidase of the Plant Plasma Membrane Has Properties of a NADH:Protein Disulfide Reductase

Plasma membranes of plant cells are characterized by a plant hormone (auxin)-responsive oxidation of NADH. The latter proceeds under argon. Also, when NADH oxidation is stimulated 50% by auxin addition, oxygen consumption is reduced by 40%. These findings are reconciled by direct assays using 5,5'-dithiobis-(2nitrobenzoic acid) (DTNB) (Ellman's reagent) that show protein disulfides to be electron acceptors for auxin-stimulated NADH oxidation. In the presence of an external reducing agent such as NADH, cysteine, or dithiothreitol, protein disulfides of the membrane are reduced with a concomitant stoichiometric increase in free thiols. In the absence of an external reducing agent, or in the presence of oxidized glutathione, DTNB-reactive thiols of the plasma membrane are decreased in the presence of auxins. Several auxin-reductant combinations were effective, but the same reductants plus chemically related and growth-inactive auxin analogs were not. A cell surface location of the affected thiols demonstrated with detergents and impermeant thiol reagents suggests that the protein may have a different physiological role than oxidation of NADH. For example, it may carry out some other role more closely related to the function of the auxin hormones in cell enlargement such as protein disulfide-thiol interchange.

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.

Glutamine transport by vesicles isolated from tumour-cell mitochondrial inner membrane.

Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.

Effects of membrane-permeant and -impermeant thiol reagents on Ca2+ and K+ channel currents of mouse pancreatic B cells

The membrane permeant thiol reagent diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) has been shown to inhibit insulin secretion and Ca2+ uptake in pancreatic B cells in the presence of a stimulating glucose concentration (20 mM), whereas the nonpenetrating analog of DIP (bis-N'-methyliodide; DIP + 2) stimulates insulin release and Ca2+ uptake at a low glucose concentration (3 mM). The effects of DIP and DIP + 2 were tested on currents through ATP-sensitive K+ (K+ATP) channels and voltage-dependent Ca2+ channels (with Ba2+ as the charge carrier) in mouse pancreatic B cells in the whole-cell mode of the patch-clamp technique. DIP (0.1 mM) almost completely inhibited both the K+ATP and Ca2+ channel currents. In contrast, DIP + 2 (0.1 mM) did not affect the Ca2+ channel current but reduced the whole-cell K+ATP current by about 40%. The data strongly suggest that the suppression of insulin secretion previously observed with DIP is due to a reduction of the current through voltage-dependent Ca2+ channels, whereas the stimulation of hormone release induced by DIP + 2 is caused by the partial inhibition of K+ATP channel current.

Effects of membrane-permeant and -impermeant thiol reagents on Ca2+ and K+ channel currents of mouse pancreatic B cells.

The membrane permeant thiol reagent diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) has been shown to inhibit insulin secretion and Ca2+ uptake in pancreatic B cells in the presence of a stimulating glucose concentration (20 mM), whereas the nonpenetrating analog of DIP (bis-N'-methyliodide; DIP + 2) stimulates insulin release and Ca2+ uptake at a low glucose concentration (3 mM). The effects of DIP and DIP + 2 were tested on currents through ATP-sensitive K+ (K+ATP) channels and voltage-dependent Ca2+ channels (with Ba2+ as the charge carrier) in mouse pancreatic B cells in the whole-cell mode of the patch-clamp technique. DIP (0.1 mM) almost completely inhibited both the K+ATP and Ca2+ channel currents. In contrast, DIP + 2 (0.1 mM) did not affect the Ca2+ channel current but reduced the whole-cell K+ATP current by about 40%. The data strongly suggest that the suppression of insulin secretion previously observed with DIP is due to a reduction of the current through voltage-dependent Ca2+ channels, whereas the stimulation of hormone release induced by DIP + 2 is caused by the partial inhibition of K+ATP channel current.

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.

Energy-dependent variation of thiol groups reactivity or accessibility in rat liver mitochondria, revealed by measurements of labelled thiol reagents incorporation

Permeant and impermeant labelled thiol reagents were incubated with rat liver mitochondria, and incorporation of reagent into mitochondria estimated. With permeant thiol reagents, incorporation depends on the energetic state of mitochondria; when coupled electron-transfer takes place, incorporation is fairly increased; the stimulation is abolished in the presence of an uncoupler, an electron-transfer inhibitor or inorganic phosphate. With an impermeant thiol reagent, the incorporation is unaffected by the energetic state of the mitochondria. These results favour the view of a participation of thiol groups in the energy-conserving mechanism but it cannot be ruled out that part of the unmasked thiol groups are implicated in the phosphate transport system. The observed stimulation may reflect either an increase in accessibility or in reactivity of some mitochondrial thiol groups.

Involvement of thiol function in the activity of energy transfer factor D of mitochondrial oxidative phosphorylation

Permeant and impermeant labelled thiol reagents were incubated with rat liver mitochondria, and incorporation of reagent into mitochondria estimated. With permeant thiol reagents, incorporation depends on the energetic state of mitochondria; when coupled electron-transfer takes place, incorporation is fairly increased; the stimulation is abolished in the presence of an uncoupler, an electron-transfer inhibitor or inorganic phosphate. With an impermeant thiol reagent, the incorporation is unaffected by the energetic state of the mitochondria. These results favour the view of a participation of thiol groups in the energy-conserving mechanism but it cannot be ruled out that part of the unmasked thiol groups are implicated in the phosphate transport system. The observed stimulation may reflect either an increase in accessibility or in reactivity of some mitochondrial thiol groups.