Inside-out Membrane Vesicles Research Articles

GSH Inhibits Trypsinization of the C-terminal Half of Human MRP1

MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance to tumor cells. The accumulated evidence has proved that GSH interacts with MRP1 and stimulates drug transport. However, the mechanism of GSH-dependent drug transport by MRP1 remains unclear. In this study, we used limited tryptic digestion of MRP1 in isolated membrane vesicles, in the presence and absence of GSH, to investigate the influence of GSH on MRP1 conformation. We found that GSH inhibited the generation of an approximately 35-kDa C-terminal tryptic fragment (including a C-terminal His tag) termed C2 from MRP1. This effect of GSH was not because of direct inhibition of trypsin activity, and agosterol A enhanced the inhibitory effect of GSH. The main cleavage site in MRP1 for the generation of the C2 fragment by trypsin resided between TMD2 and NBD2 of MRP1. Limited tryptic digestion of membrane vesicles expressing various truncated and co-expressed MRP1 fragments in the presence and absence of GSH revealed that GSH inhibited the production of the C2 fragment only in the presence of the L(0) region of MRP1. Thus the L(0) region is required for the inhibition of trypsinization of the C-terminal half of MRP1 by GSH. These findings, together with previous reports, suggest that GSH induces a conformational change at a site within the MRP1 that is indispensable for the interaction of MRP1 with its substrates.

The First Cytoplasmic Loop of the Mannitol Permease from Escherichia coli is Accessible for Sulfhydryl Reagents from the Periplasmic Side of the Membrane

The mannitol permease (EII(Mtl)) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII(Mtl) contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII(Mtl), all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII(Mtl) undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.

Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.

Binding affinity of lactose permease is not altered by the H+ electrochemical gradient.

The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated that the binding site has alternating access to either side of the membrane during turnover. Here, the affinity of LacY for ligand in right-side-out or inside-out membrane vesicles is measured in the absence or presence of an H(+) electrochemical gradient (Deltamicro(H(+))) by utilizing ligand protection against alkylation. Right-side-out or inside-out membrane vesicles containing LacY with a single cysteine residue at position 148 exhibit K(D) values for lactose or beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside of approximately 1.0 mM or 40 microM, respectively, and no systematic change is observed in the presence of Deltamicro(H(+)) under conditions in which there is little or no accumulation of ligand. The results are consistent with a mechanism in which the major effect of Deltamicro(H(+)) on sugar accumulation is caused by an increased rate of deprotonation on the inner face of the membrane, leading to an increase in the rate of return of the unloaded symporter to the outer face of the membrane.

The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine.

Recently, mutations in the novel polytopic integral membrane protein PfCRT were shown to cause chloroquine resistance (CQR) in the malarial parasite Plasmodium falciparum. PfCRT is not a member of the well-known family of ABC proteins that have previously been associated with other drug resistance phenomena. Thus, the mechanism(s) whereby mutant PfCRT molecules confer antimalarial drug resistance is (are) unknown. Previously, we succeeded in overexpressing PfCRT to high levels in Pichia pastoris yeast by synthesizing a codon-optimized version of the pfcrt gene. Using purified membranes and inside-out plasma membrane vesicles (ISOV) isolated from strains harboring either wild-type or CQR-associated mutant PfCRT, we now show that under deenergized conditions the PfCRT protein specifically binds the antimalarial drug chloroquine (CQ) with a K(D) near 400 nM but does not measurably bind the related drug quinine (QN) at physiologically relevant concentrations. Transport studies using ISOV show that QN is passively accumulated as expected on the basis of previous measurement of the ISOV DeltapH for the different strains. However, passive accumulation of CQ is lower than expected for ISOV harboring mutant PfCRT, despite higher DeltapH for these ISOV.

Unconventional Secretion of Fibroblast Growth Factor 2 Is Mediated by Direct Translocation across the Plasma Membrane of Mammalian Cells

Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system.

Transport of ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins MRP1, MRP2, and MRP3.

Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [(3)H]EE-G was observed in MRP2 (K(m) of 35.1 +/- 3.5 microM) and MRP3 (K(m) of 9.2 +/- 2.3 microM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17beta-estradiol-17beta-D-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.

Membrane Targeting of a Bacterial Virulence Factor Harbouring an Extended Signal Peptide

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.

Naproxen affects Ca 2+ fluxes in mitochondria, microsomes and plasma membrane vesicles

There is substantial evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) affect cellular processes regulated by Ca 2+ ions, including the metabolic responses of the liver to Ca 2+-dependent hormones. The aim of the present study was to determine whether the effects of naproxen are mediated by a direct action on cellular Ca 2+ fluxes. The effects of naproxen on 45 Ca 2+ fluxes in mitochondria, microsomes and inside-out plasma membrane vesicles were examined. Naproxen strongly impaired the mitochondrial capacity to retain 45 Ca 2+ and inhibited also ATP-dependent 45 Ca 2+ uptake by microsomes. Naproxen did not modify 45 Ca 2+ uptake by inside-out plasma membrane vesicles, but it inhibited the hexokinase/glucose-induced Ca 2+ efflux from preloaded vesicles. Additional assays performed in isolated mitochondria revealed that naproxen causes mitochondrial uncoupling and swelling in the presence of Ca 2+ ions. These effects were prevented by EGTA, ruthenium red and cyclosporin A, indicating that naproxen acts synergistically with Ca 2+ ions by promoting the mitochondrial permeability transition. The experimental results suggest that naproxen may impair the metabolic responses to Ca 2+-dependent hormones acting by at least two mechanisms: (1) by interfering with the supply of external Ca 2+ through a direct action on the plasma membrane Ca 2+ influx, and (2) by affecting the refilling of the agonist-sensitive internal stores, including endoplasmic reticulum and mitochondria.

Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

CGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.

Identification of Domains Participating in the Substrate Specificity and Subcellular Localization of the Multidrug Resistance Proteins MRP1 and MRP2

The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C4 (LTC4) (Km = 0.1 and 1 microm). To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells. We examined the kinetic properties of each chimeric protein by measuring LTC4 and methotrexate transport in inside-out membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods. The following results were determined in these studies: (i) when the NH2-proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, Km(LTC4) values of the chimera decreased approximately 4-fold and Km(methotrexate) values increased approximately 5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased approximately 3-fold; (ii) when the NH2-proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins. These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC4 and anticancer agents.

Differential Modulation of the Human Liver Conjugate Transporters MRP2 and MRP3 by Bile Acids and Organic Anions

The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17-beta-d-glucuronide (E217betaG), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E217betaG transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E217betaG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217betaG, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.

Characterization of the MRP4- and MRP5-mediated Transport of Cyclic Nucleotides from Intact Cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Atp Binding Cassette Multidrug Transporters Limit the Anti-HIV Activity of Zidovudine and Indinavir in Infected Human Macrophages

To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5.

The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.

In Vitro Synthesis of Lactose Permease to Probe the Mechanism of Membrane Insertion and Folding

Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein from Escherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N(6)) and probe changes in conformation during insertion into the membrane. Specifically, when N(6) remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N(6) is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N(6) is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N(6) is degraded by FtsH protease in vivo, and N(6) with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N(6) remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N(6) and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains.

Bioflavonoid stimulation of glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1).

In tumor cells, the human multidrug resistance protein 1 (MRP1), confers resistance to a broad spectrum of anticancer agents. MRP1 is also expressed in many normal tissues where it acts as an ATP-dependent transporter of organic anions. Reduced glutathione (GSH) is transported by MRP1 with very low affinity, and certain MRP1 substrates are transported in association with this tripeptide. Previous studies have shown that various dietary flavonoids stimulate the ATPase activity of MRP1 and inhibit transport of its conjugated organic anion substrates but are poor reversers of MRP1-mediated drug resistance. In contrast, many of the same flavonoids markedly stimulate GSH transport by MRP1. In the present study, we found that stimulation of GSH transport in inside-out MRP1-enriched membrane vesicles by apigenin, naringenin, genistein, and quercetin was maximum at a concentration of 30 microM. Apigenin was the most efficacious of the four bioflavonoids, showing a maximal 6-fold increase over basal levels of GSH transport. The apparent K(m) and V(max) for GSH uptake in the presence of 30 microM apigenin were 116 microM and 666 pmol mg(-1) min(-1), respectively. Chemosensitivity assays with control-transfected and MRP1-transfected HeLa cell lines showed that the IC(50) values for apigenin, naringenin, genistein, and quercetin were similar, demonstrating that overexpression of MRP1 does not confer resistance to these bioflavonoids. Our results suggest that flavonoids stimulate MRP1-mediated GSH transport by increasing the apparent affinity of the transporter for GSH but provide no evidence that a cotransport mechanism is involved.

The ATPase Activity and the Functional Domain of PotA, a Component of the Spermidine-preferential Uptake System inEscherichia coli

The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, -B, -C, and -D, was studied using purified PotA and a PotABC complex on inside-out membrane vesicles. It was found that PotA can form a dimer by disulfide cross-linking but that each PotA molecule functions independently. When PotA was associated with the membrane proteins PotB and PotC, the K(m) value for ATP increased and PotA became much more sensitive to inhibition by spermidine. It was also shown that spermidine uptake in cells was gradually inhibited in parallel with spermidine accumulation in cells. The results suggest that spermidine functions as a feedback inhibitor of spermidine transport. The function of PotA was analyzed using PotA mutants obtained by random mutagenesis. There are two domains in PotA. The NH2-terminal domain (residues 1-250) contains the ATP binding pocket formed in part by residues Cys26, Phe27, Phe45, Cys54, Leu60, and Leu76, the active center of ATPase that includes Val135 and Asp172, and amino acid residues necessary for the interaction with a second PotA subunit (Cys26) and with PotB (Cys54). The COOH-terminal domain (residues 251-378) of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity.

Separate Analysis of Twin-arginine Translocation (Tat)-specific Membrane Binding and Translocation in Escherichia coli

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H(+) gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.