Inside-out Membrane Vesicles Research Articles

A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.

The identification of a new family of sugar efflux pumps in Escherichia coli.

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radiodurans, appear to encode closely related proteins. An in vitro transport assay using inside-out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]-lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.

Effects of ion gradients on H+ transport mediated by human MDR 1 protein.

In the previous paper we presented a variety of data consistent with significant perturbations in 9.3 yeast plasma membrane ion transport upon overexpression of the hu MDR 1 protein. Thus, in this paper, we compare formation of DeltapH for inside-out yeast plasma membrane vesicles (ISOV) prepared from control 9.3/pVT versus 9.3/hu MDR 1 yeast. Since MDR 1 ATPase activity has a broader, more alkaline pH profile relative to endogenous yeast H+ ATPase activity, we analyzed H+ pumping at pH >/= 8.0 in detail in order to selectively amplify hu MDR 1 contributions to H+ movement over those of the endogenous yeast H+ ATPase. We observed: (1) imposition of a Cl- gradient oriented outside to in enhances acidification for 9.3/pVT ISOV (as expected), but decreases acidification for 9.3/hu MDR 1 ISOV; (2) imposition of a Cl- gradient oriented inside to out decreases acidification for 9.3/pVT ISOV (as expected) but enhances acidification for 9.3/hu MDR 1 ISOV; (3) a Na+ gradient oriented in the same direction as the Cl- gradient amplifies the effects due to hu MDR 1 when both gradients are oriented inside to out, but not outside to in. The data are most easily explained by interesting Na+, Cl-, and ATP-dependent H+ transport mediated by hu MDR 1 protein as previously suggested [Hoffman and Roepe (1997) Biochemistry 36, 11153-11168]. These data may help to resolve a variety of conflicting reports in the literature regarding ion transport mediated by hu MDR 1 and have implications for the physiology of a number of polarized epithelia in which hu MDR 1 is endogenously expressed.

Evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein.

Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for verapamil stimulation of MDR 1 ATPase activity and H+ pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes H+ transport in some fashion and lowers membrane potential in yeast when K+ contributes strongly to that potential. In the accompanying paper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined.

ATP-dependent calcium uptake into basolateral vesicles from transporting epithelia of intermolt crayfish.

ATP-dependent Ca2+ uptake was determined into inside-out basolateral membrane vesicles (BLMV) from intermolt crayfish (Procambarus clarkii) Ca2+-transporting epithelia: gill, hepatopancreas (liver), and antennal gland (kidney). Extravesicular (EV) ATP (5 mM) increased 45Ca2+ uptake (free Ca2+ 5 microM) by fivefold but was abolished by pretreatment with either vanadate or the ionophore A-23187. Addition of A-23187 to Ca2+-loaded vesicles produced 70% efflux. The saturable carrier exhibited a Km for Ca2+ of 0.11-0.27 microM and maximal influx of 20-123 pmol. mg-1. min-1. The Km for ATP was 0.01-0.04 mM. The temperature coefficient ranged from 1.43 to 2.06. EGTA treatment of hepatopancreas and antennal gland vesicles decreased 45Ca2+ uptake by 50-90%; uptake was restorable by calmodulin. However, in gill, 45Ca2+ uptake was unaffected by EGTA treatment and calmodulin decreased uptake in both EGTA-treated and untreated vesicles. Addition of EV Na+ (5 mM) increased ATP-dependent Ca2+ uptake into hepatopancreas and antennal gland BLMV by 60%; in hepatopancreas BLMV, this increase was inhibitable by ouabain. However, ATP-dependent Ca2+ uptake in gill vesicles was Na+ independent. The relative role of each epithelium in whole animal Ca2+ homeostasis has been interpreted based on in vitro characteristics.

Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP.

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease.

Previously we presented evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone in the folding of the polytopic membrane protein lactose permease (LacY) of Escherichia coli. Here we provide more definitive evidence supporting the chaperone properties of PE. Membrane insertion of LacY prevents its irreversible aggregation, and PE participates in a late step of conformational maturation. The temporal requirement for PE was demonstrated in vitro using a coupled translation-membrane insertion assay that allowed the separation of membrane insertion from phospholipid-assisted folding. LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antibodies, when synthesized in the presence of PE-containing inside-out membrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-translationally. The presence of IOVs lacking PE and containing anionic phospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively. Therefore, critical folding steps occur after membrane insertion dependent on the interaction of LacY with PE to prevent illicit interactions which lead to misfolding of LacY.

Transport of arachidonic acid across the neutrophil plasma membrane via a protein-facilitated mechanism.

Arachidonic acid is the rate-limiting substrate in the biosynthesis of leukotrienes in activated neutrophils. Liberation of arachidonate from intracellular membranes and uptake of exogenous arachidonate are the two principal mechanisms by which the cell can increase the level of this substrate. We investigated arachidonate uptake and export by using intact polymorphonuclear neutrophils and inside-out plasma membrane vesicles thereof. Here we show that the cellular uptake of arachidonate is energy dependent with an energy of activation (EA) of 10.0 kcal/mol and half-saturated at an arachidonate concentration of 4.8 nmol/mg of cell protein. Protein-facilitated transport of arachidonate across the plasma membrane in either direction is sensitive to proteases, chemical protein modifying reagents, anion transport inhibitors, and, most notably, toward several structurally unrelated leukotriene B4 receptor antagonists with IC50 values in the range of 16-44 microM. The inhibitors did not inhibit the diffusional uptake of methyl arachidonate into neutrophils and inside-out plasma membrane vesicles, indicating that a transport protein is required for the rapid uptake of the free acid but not for the uptake of the ester. Other long-chain fatty acids did compete with the uptake of arachidonate in both assay systems, whereas leukotriene B4 did not. This study documents a novel protein-facilitated transport mechanism for arachidonate in neutrophils, potentially involved in transcellular eicosanoid biosynthesis and sPLA2-mediated arachidonate signaling in neutrophils.

Dissection of drug‐binding‐induced conformational changes in P‐glycoprotein

P-glycoprotein (Pgp) is a membrane-transport ATPase. It uses energy from ATP hydrolysis to transport pleiotropic cytotoxic drugs from inside to outside of cells. Thus, elevated expression of Pgp in cancer cells causes multidrug resistance. It is now known that the conformational state of Pgp changes during its catalytic cycle. However, how ATP hydrolysis relates to drug binding by Pgp is yet to be determined. In this study, we used limited trypsin digestion of Pgp in isolated inside-out membrane vesicles to investigate the effects of drugs on Pgp conformation and to determine the drug-bound conformational states of Pgp in the catalytic cycle. We found that (a) binding of vinblastine or verapamil alone can cause a conformational change in Pgp, but the change induced by the drug binding is different from that induced by nucleotide binding, (b) there may be at least two binding sites for Pgp substrates, one for drugs such as vinblastine and verapamil and the other for drugs such as colchicine and adriamycin, (c) the conformation of Pgp bound by ATP and vinblastine is different from the conformation bound by either one alone, and (d) the ADP-bound Pgp does not bind vinblastine. Based on these observations and our previous studies, we propose a model for drug binding and transport in the catalytic cycle of Pgp.

Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs)

The effect on cytotoxicity of combining a range of clinically important non-steroidal anti-inflammatory drugs (NSAIDs) with a variety of chemotherapeutic drugs was examined in the human lung cancer cell lines DLKP, A549, COR L23P and COR L23R and in a human leukaemia line HL60/ADR. A specific group of NSAIDs (indomethacin, sulindac, tolmetin, acemetacin, zomepirac and mefenamic acid) all at non-toxic levels, significantly increased the cytotoxicity of the anthracyclines (doxorubicin, daunorubicin and epirubicin), as well as teniposide, VP-16 and vincristine, but not the other vinca alkaloids vinblastine and vinorelbine. A substantial number of other anticancer drugs, including methotrexate, 5-fluorouracil, cytarabine, hydroxyurea, chlorambucil, cyclophosphamide, cisplatin, carboplatin, mitoxantrone, actinomycin D, bleomycin, paclitaxel and camptothecin, were also tested, but displayed no synergy in combination with the NSAIDs. The synergistic effect was concentration dependent. The effect appears to be independent of the cyclo-oxygenase inhibitory ability of the NSAIDs, as (i) the synergistic combination could not be reversed by the addition of prostaglandins D 2 or E 2; (ii) sulindac sulphone, a metabolite of sulindac that does not inhibit the cyclooxygenase enzyme, was positive in the combination assay: and (iii) many NSAIDs known to be cyclo-oxygenase inhibitors, e.g. meclofenamic acid, diclofenac, naproxen, fenoprofen, phenylbutazone, flufenamic acid, flurbiprofen, ibuprofen and ketoprofen, were inactive in the combination assay. The enhancement of cytotoxicity was observed in a range of drug sensitive tumour cell lines, but did not occur in P-170-overexpressing multidrug resistant cell lines. However, in the HL60/ADR and COR L23R cell lines, in which multidrug resistance is due to overexpression of the multidrug resistance-associated protein MRP, a significant increase in cytotoxicity was observed in the presence of the active NSAIDs. Subsequent Western blot analysis of the drug sensitive parental cell lines, DLKP and A549, revealed that they also expressed MRP and reverse-transcription–polymerase chain reaction studies demonstrated that mRNA for MRP was present in both cell lines. It was found that the positive NSAIDs were among the more potent inhibitors of [ 3H]-LTC 4 transport into inside-out plasma membrane vesicles prepared from MRP-expressing cells, of doxorubicin efflux from preloaded cells and of glutathione-S-transferase activity. The NSAIDs did not enhance cellular sensitivity to radiation. The combination of specific NSAIDs with anticancer drugs reported here may have potential clinical applications, especially in the circumvention of MRP-mediated multidrug resistance.

P1G183 - Comparative efficacy of I.V. Pralidoxime vs. NaHCO 3 in rats lethally poisoned with O-P insecticide

2, 3-Dimercaptopropane-1-sulfonic acid (DMPS) and meso-2, 3-Dimercaptosuccinic acid (DMSA) are dithiols used to treat humans exposed to methylmercury (CH3Hg+). After treatment, significant amounts of mercury are eliminated rapidly from the kidneys and are excreted in urine. In the present study, we extended our previous studies by testing the hypothesis that MRP2 mediates the secretion of DMPS or DMSA S-conjugates of CH3Hg+. To test this hypothesis, the disposition of mercury was assessed in control and Mrp2-deficient (TR−) rats exposed intravenously to a 5.0-mg/kg dose of CH3HgCl. Twenty-four and 28 h after exposure, groups of four control and four TR− rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h later. Renal and hepatic contents of mercury were greater in saline-injected TR− rats than in controls. In contrast, the amounts of mercury excreted in urine and feces by TR− rats were less than those by controls. DMPS and DMSA significantly reduced the renal and hepatic content of mercury in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of mercury (which was greater in the controls) was also observed. Our findings in inside-out membrane vesicles prepared from hMRP2-transfected Sf9 cells show that uptake of DMPS and DMSA S-conjugates of CH3Hg+ was greater in the vesicles containing hMRP2 than in control vesicles. Overall, these dispositional findings indicate that MRP2 does play a role in DMPS- and DMSA-mediated elimination of mercury from the kidney.

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 m M ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.

Requirements for the Translocation of Elongation-arrested, Ribosome-associated OmpA across the Plasma Membrane ofEscherichia coli

An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli. In this way, NH2-terminal pOmpA fragments of distinct sizes were synthesized. Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosome-associated nascent chains. Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E. coli required SecA and (depending on size) SecB. Whereas their translocation was strictly dependent on the H+-motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained. SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles. However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome. These results indicate that in E. coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.

Characterization of the pH dependence of hemoglobin binding to band 3: Evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex

The pH dependence of hemoglobin binding to inside-out red cell membrane vesicles was studied using 90° light scattering (Salhany, J.M. et al., Biochemistry 19 (1980) 1447–1454). Hyperbolic binding curves were observed for high-affinity hemoglobin binding to the cytoplasmic domain of band 3 (CDB3) within the intact transporter. Analysis of these saturation curves yielded the apparent K d and the maximum light scattering signal change (ΔLS max ). The apparent K d for hemoglobin binding did not change substantially between pH 5.5 and 7.0, while at pH 8, there is no binding. In contrast, ΔLS max decreased by about 20-fold between pH 5.5 and 7.0, with an apparent p K of 6.5. These results suggest that hemoglobin binds to CDB3 with high affinity at both neutral and acid pH, a suggestion that was confirmed using a centrifugation method. Thus, the p K for the light scattering signal change is significantly lower than the p K for the actual binding process. We show that the change in ΔLS max is not related to a change in band 3 binding capacity, which remains constant as a function of pH. We also show that hemoglobin binding to non-band 3 sites contributes less than 10% to ΔLS max under our specific experimental conditions. On the basis of these and previous fluorescence quenching results in the literature, we propose a new model for hemoglobin binding to band 3, where raising the pH from 6 and 7 causes the CDB3–hemoglobin complex to undergo a conformational change leading to the movement of the bound hemoglobin away from the surface of the bilayer. The possible implication of this new mechanistic interpretation is discussed briefly.

Human mast cells secreting leukotriene C4 express the MRP1 gene-encoded conjugate export pump.

Mast cells are known to secrete endogenously synthesized leukotriene C4 (LTC4), but the identity of the responsible export pump in human mast cells was unknown. The multidrug resistance proteins MRP1 and MRP2 have been identified as primary-active ATP-dependent export pumps for various amphiphilic anions including the glutathione conjugate LTC4. We therefore studied the expression at the RNAand protein levels of both MRP1 and MRP2 as well as the ATP-dependent LTC4 transport in the human mast cell line HMC-1. Upon stimulation by 1 microM ionomycin, intact HMC-1 cells generated 26 pmol LTC4/10(8) cells within 20 min. Transport experiments using inside-out HMC-1 membrane vesicles demonstrated an ATP-dependent LTC4 transport amounting to 1.4 pmol x (mg protein)(-1) x min(-1). Reverse transcription PCR indicated that HMC-1 cells express mRNA of MRP1, but not of MRP2 or MRP3. Cloning and sequencing of the amplified PCR fragment confirmed its identity with the human MRP1 sequence. Immunoblots using antibodies against MRP1 and MRP2 demonstrated that HMC-1 cells contain the MRP1 but not the MRP2 protein. Our results indicate that the 190 kDa integral membrane glycoprotein MRP1 mediates the ATP-dependent export of LTC4 from human mast cells to the extracellular space.

Conformational changes of P-glycoprotein by nucleotide binding

P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.

Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein.

P-glycoprotein is an ATP-dependent plasma membrane multidrug transporter of broad specificity. A common chemical property of its substrates is that all are lipophilic. Using Hoechst 33342 as the substrate, we have previously shown that P-glycoprotein extracts the substrate directly from the lipid bilayer [Shapiro, A. B., Corder, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 115-121]. In this paper, we determined the leaflet of the plasma membrane from which P-glycoprotein extracts Hoechst 33342. The initial rate of Hoechst 33342 transport upon ATP addition to P-glycoprotein-rich inside-out plasma membrane vesicles decreased slightly with the amount of time previously elapsed for slow diffusion of Hoechst 33342 to the extracellular leaflet. This result is consistent with transport from the cytoplasmic leaflet. Fluorescence resonance energy transfer from donor Hoechst 33342 to acceptor 2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero- 3-phosphocholine (Nbd-C6-HPC) in the cytoplasmic leaflet was used to monitor the amount of Hoechst 33342 in the cytoplasmic leaflet versus time. The initial rate of decrease of the energy-transfer-related Nbd-C6-HPC fluorescence after ATP addition exceeded that of the Hoechst 33342 fluorescence and continued to decrease after decrease of the Hoechst 33342 fluorescence had ceased. These effects were consistent with transport of Hoechst 33342 from the cytoplasmic leaflet to the aqueous interior of the vesicles, followed by rebinding to the extracellular leaflet. This demonstrates that P-glycoprotein transports drugs from the cytoplasmic leaflet of the plasma membrane directly to the aqueous extracellular medium. This finding has implications for efforts to localize the drug-binding site(s) within P-glycoprotein.

Topology Mapping of the Amino-terminal Half of Multidrug Resistance-associated Protein by Epitope Insertion and Immunofluorescence

The multidrug resistance-associated protein (MRP) is an integral membrane protein that causes multidrug resistance when overexpressed in mammalian cells. Within the ATP-binding cassette superfamily, MRP belongs to a subgroup of structurally and functionally related proteins that includes the yeast cadmium factor 1 and yeast oligomycin resistance I proteins, and the mammalian sulfonylurea receptors SUR1 and SUR2. Hydropathy analysis of these proteins predicts a unique membrane-associated region at the amino terminus followed by a structural unit composed of 12 transmembrane (TM) domains and two nucleotide-binding domains that is characteristic of eukaryotic ATP-binding cassette transporters. The topology of the membrane-associated regions of MRP remains largely unknown and was investigated. Small hemagglutinin epitopes (YPYDVPDYAS) were inserted in predicted hydrophilic segments of the membrane-associated regions from the amino-terminal half of MRP and these proteins were expressed in HeLa cells, and tested for their capacity to confer etoposide resistance. The polarity of the inserted tags with respect to plasma membrane was then deduced by immunofluorescence in intact and permeabilized cells. Insertion of epitopes at positions 4, 163, 271, 574, and 653 produced functional proteins while insertions at positions 127, 417, 461, and 529 abrogated the capacity of MRP to confer drug resistance. Epitopes inserted at positions 4, 163, and 574 were localized extracellularly, whereas those inserted at positions 271 and 653 revealed an intracellular location. Although a single epitope inserted at position 461 was compatible with MRP function, it was inaccessible to the anti-epitope antibody and two copies of the tag at that site abrogated MRP function. These results indicate that the amino terminus of MRP is extracellular, while the linker segment joining the first and second membrane-associated regions is intracellular as is the first nucleotide-binding domain. Our findings are therefore consistent with a topological model of MRP that contains 5 TM segments in the first membrane-associated region and 6 TM segments in the second membrane region.

Characterization of a Diamine Exporter in Chinese Hamster Ovary Cells and Identification of Specific Polyamine Substrates

Export of the diamine putrescine was studied using inside-out plasma membrane vesicles prepared from Chinese hamster cells. Putrescine uptake into vesicles was a saturable and an ATP- and antizyme-independent process. Excess amounts of a series of diamines or monoacetyl spermidine, but not monoacetyl putrescine, spermidine, or spermine, inhibited putrescine transport. Putrescine uptake into vesicles prepared at pH 7.4 was suppressed at pH 5, compared with pH 7.4; was stimulated approximately 2.5-fold at pH 7.4 in vesicles prepared at pH 6.25, compared with vesicles prepared at pH 7.4; and was not inhibited by valinomycin in the presence of potassium ions. Reserpine and verapamil blocked [3H]putrescine uptake into inverted vesicles. Verapamil treatment caused an increase in intracellular contents of putrescine, cadaverine, and N8-acetylspermidine, in unstressed proliferating cells, or of N1-acetylspermidine, in cells subjected to heat shock to induce acetylation of spermidine at N1. These data indicate that putrescine export in Chinese hamster cells is mediated by a non-electrogenic antiporter capable of using protons as the counter ion. Physiological substrates for this exporter include putrescine, cadaverine, and monoacetyl spermidine and have the general structure NH3+-(CH2)n-NH2 + R at acidic or neutral pH.

Protein-facilitated Export of Arachidonic Acid from Pig Neutrophils

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.