ATP-binding cassette (ABC) transporters mediate nonvesicular, raft-modulated sterol movement from the plasma membrane to the endoplasmic reticulum

Little is known about the mechanisms of intracellular sterol transport or how cells maintain the high sterol concentration of the plasma membrane (PM). Here we demonstrate that two inducible ATP-binding cassette (ABC) transporters (Aus1p and Pdr11p) mediate nonvesicular movement of PM sterol to the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. This transport facilitates exogenous sterol uptake, which we find requires steryl ester synthesis in the ER. Surprisingly, while expression of Aus1p and Pdr11p significantly increases sterol movement from PM to ER, it does not alter intracellular sterol distribution. Thus, ER sterol is likely rapidly returned to the PM when it is not esterified in the ER. We show that the propensity of PM sterols to be moved to the ER is largely determined by their affinity for sterol sphingolipid-enriched microdomains (rafts). Our findings suggest that raft association is a primary determinant of sterol accumulation in the PM and that Aus1p and Pdr11p facilitate sterol uptake by increasing the cycling of sterol between the PM and ER.     

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.     

Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake

The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL-C) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.     

Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters

The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.     

A novel ligand bound ABC transporter, LolCDE, provides insights into the molecular mechanisms underlying membrane detachment of bacterial lipoproteins

The LolCDE complex of Escherichia coli belongs to the ABC transporter superfamily and initiates the lipoprotein sorting to the outer membrane by catalysing their release from the inner membrane. LolC and/or LolE, membrane subunits, recognize lipoproteins anchored to the outer surface of the inner membrane, while LolD hydrolyses ATP on its inner surface. We report here that ligand-bound LolCDE can be purified from the inner membrane in the absence of ATP. Liganded LolCDE represents an intermediate of the release reaction and exhibits higher affinity for ATP than the unliganded form. ATP binding to LolD weakens the interaction between LolCDE and lipoproteins and causes their dissociation in a detergent solution, while lipoprotein release from membranes requires ATP hydrolysis. Liganded LolCDE thus reveals molecular events brought about through ATP binding and hydrolysis. LolCDE is the first example of an ABC transporter purified with tightly bound native substrates. A single molecule of lipoprotein is found to bind per molecule of the LolCDE complex.     

Functional Rescue of a Misfolded Eukaryotic ATP-binding Cassette Transporter by Domain Replacement

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.     

Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein

Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium.     

The effects of altered sterol composition on the mitochondrial adenine nucleotide transporter of Saccharomyces cerevisiae.

1. The membrane sterol composition of mitochondria of the ole-3 mutant of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of Tween 80 (1%, W/V) plus defined supplements o- delta-aminolaevulinate. 2. Changes in mitochondrial sterol content induced considerable changes in the adenine nucleotide transporter. 3. As the sterol content was decreased, the affinity of the transporter for ATP did not alter significantly, but the rate of ATP uptake was greatly decreased, the total number of atractylate-sensitive binding sites diminished, and the proportion of high-affinity binding sites was decreased. 4. Since sterol depletion also uncouples oxidative phosphorylation [Astin & Haslam (1977) Biochem. J., 166, 287-298] and prevents the intramitochondrial generation of ATP, the decrease in the rate of ATP uptake by sterol-depleted mitochondria will cause a decrease in intramitochondrial ATP concentrations in vivo. This probably explains the inhibition of mitochondrial macromolecular synthesis that has previously been reported in lipid-depleted yeast mitochondria.     

The calcium-transporting ATPase and the calcium- or magnesium-dependent nucleotide phosphatase activities of human placental trophoblast basal plasma membrane are separate enzyme activities

The properties of calcium-stimulated ATP hydrolysis often differ from those of ATP-dependent calcium transport. We have characterized two components of calcium-stimulated ATP hydrolysis in human placental basal plasma membrane. In the absence of magnesium, component 1 apparently has saturable sites for free calcium in both the nanomolar and low micromolar range. It was stimulated by either calcium or magnesium, was unselective for nucleotide substrate, and its activity was very much greater than that of ATP-dependent calcium transport. Component 1 was inhibited by GTP, permitting measurement of component 2 with activity and magnesium stimulation comparable to ATP-dependent calcium transport. Component 2 was inhibited partially by an antibody against purified erythrocyte calcium transporter and completely by sulfhydryl reagents, whereas component 1 was unaffected. A phosphorylated intermediate of the calcium transporter co-migrated with the erythrocyte transporter on acidic sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Immunostaining after transfer to nitrocellulose revealed a doublet. The band of lower molecular weight co-migrated with that of the human erythrocyte membrane transporter. The addition of GTP permits separate measurement of ATP hydrolysis by the calcium transporter of the placental basal plasma membrane and may be useful in defining its properties in other cell membranes under a variety of conditions.     

ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells

Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells.     

In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.     

Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

Energy Coupling Efficiency in the Type I ABC Transporter GlnPQ

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Förster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.     

Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism

ATP-binding cassette (ABC) transporters are integral membrane proteins that move diverse substrates across cellular membranes. ABC importers catalyse the uptake of essential nutrients from the environment, whereas ABC exporters facilitate the extrusion of various compounds, including drugs and antibiotics, from the cytoplasm. How ABC transporters couple ATP hydrolysis to the transport reaction has long remained unclear. The recent crystal structures of four complete ABC transporters suggest that a key step of the molecular mechanism is conserved in importers and exporters. Whereas binding of ATP promotes an outward-facing conformation, the release of the hydrolysis products ADP and phosphate promotes an inward-facing conformation. This basic scheme can in principle explain ATP-driven drug export and binding protein-dependent nutrient uptake.     

Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region

The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.