ABC transporters in lipid transport

Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport. The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids. Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug. The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene. Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins. Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen. Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye. ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter. A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.     

In vitro assays of vesicular transport

Movement of proteins and lipids between the various compartments of eukaryotic cells is fundamental to the maintenance of cellular homeostasis, and an understanding of the molecular mechanisms that govern these processes remains a key goal of cell biological research. This aim has been greatly facilitated by the development of assays that recapitulate specific events in vitro. In the following article we provide an overview of some of the currently used assays that measure the movement of proteins within the exocytic and endocytic pathways, and provide a starting point for those wishing to establish their own systems to study other vesicular transport steps.     

ABA transport factors found in Arabidopsis ABC transporters

Abscisic acid (ABA) is a phytohormone that plays an important role in responses to environmental stresses as well as seed maturation and germination. Intracellular signaling by ABA has been rigorously investigated in relation to stomatal guard-cell regulation, seed germination and abiotic stress responses. However, intercellular regulation of ABA, including the molecular basis of ABA transport systems, has hardly been examined in any plant species. Based on genetic and biochemical analyses, we present evidence that one of the ATP-binding cassette (ABC) transporter genes, AtABCG25, encodes a protein that functions as an ABA exporter through the plasma membrane and is involved in the intercellular ABA signaling pathway. The ABC-type transporter is conserved in model species from E. coli to humans and is reported to transport various metabolites or signaling molecules in an ATP-dependent manner. At same time, another ABC transporter in Arabidopsis, AtABCG40, was independently reported to function as an ABA importer in plant cells. These findings strongly suggest the active control of ABA transport between plant cells, and they provide a novel impetus for examining ABA intercellular regulation.Key words: Arabidopsis, ABA, transport, ABC transporter, ABCG, transposontagged lines     

Membrane reconstitution of ABC transporters and assays of translocator function

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.     

ABC transporters involved in the transport of plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, polyphenols, quinones and many further compounds having combined structures of those groups. Physiological roles of those metabolites for plants are still under investigation, but they play, at least in part, important functions as protectants for plant bodies against herbivores and pathogens, as well as from physical stresses like ultraviolet light and heat. In order to accomplish these functions, biosyntheses and accumulation of secondary metabolites are highly regulated in a temporal and spatial manner in plant organs, where they can appropriately accumulate. In this mini-review, I introduce the mechanism of accumulation and membrane transport of these metabolites, in particular, focusing on ATP-binding cassette transporters involved.     

Glycolipid substrates for ABC transporters required for the assembly of bacterial cell-envelope and cell-surface glycoconjugates

Glycoconjugates, molecules that contain sugar components, are major components of the cell envelopes of bacteria and cover much of their exposed surfaces. These molecules are involved in interactions with the surrounding environment and, in pathogens, play critical roles in the interplay with the host immune system. Despite the remarkable diversity in glycoconjugate structures, most are assembled by glycosyltransferases that act on lipid acceptors at the cytosolic membrane. The resulting glycolipids are then transported to the cell surface in processes that frequently begin with ATP-binding cassette transporters. This review summarizes current understanding of the structure and biosynthesis of glycolipid substrates and the structure and functions of their transporters. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Dysregulation of retinoid transporters expression in body fluids of schizophrenia patients

This study aims to find the biomarkers or associated proteins in body fluids of schizophrenia patients so that we can further understand the etiology of schizophrenia. We applied proteomic technologies combining two-dimensional electrophoresis with Coomassie blue staining and mass spectrometry and identified a procedure for the clinical screening of disease-influenced body fluid proteins in two sets of samples, plasma from 19 schizophrenia patients and cerebrospinal fluid (CSF) from 35 drug-treated schizophrenic patients and 36 healthy controls. The expression of transthyretin (TTR) tetramer increased significantly in plasma of schizophrenic patients after a valid 2 months in-hospital antipsychotic treatment. Conversely, the expression of the TTR tetramer and apolipoprotein E (ApoE) was down-regulated by up to 1.68 and 3.62 times, respectively, in the CSF of schizophrenia patients compared to that of normal controls, which has not been reported previously. Considering that the TTR tetramer and ApoE are both retinoid transporters, retinoid dysfunction might be involved in the pathology of schizophrenia.     

Functions of ABC transporters in plants

ABC (ATP-binding cassette) proteins are ubiquitously found in prokaryotes and eukaryotes and generally serve as membrane-intrinsic primary active pumps. In higher plants, ABC proteins constitute a large family, grouped phylogenetically into eight clusters, subfamilies ABCA-ABCI (ABCH is not found in plants). ABC transporters shuttle substrates as diverse as lipids, phytohormones, carboxylates, heavy metals, chlorophyll catabolites and xenobiotic conjugates across a variety of biological membranes. To date, the largest proportions of characterized members have been localized to the plasma membrane and the tonoplast, with dominant implications in cellular secretion and vacuolar sequestration, but they are also found in mitochondrial, plastidal and peroxisomal membranes. Originally identified as tonoplast-intrinsic proteins that shuttle xenobiotic conjugates from the cytosol into the vacuole, thus being an integral part of the detoxification machinery, ABC transporters are now recognized to participate in a multitude of physiological processes that allow the plant to adapt to changing environments and cope with biotic and abiotic stresses.     

A high-throughput screening for phosphatases using specific substrates

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.     

Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)(2)-Rhod110 and bis-(Ala-Pro)-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC trade mark collection and natural product extracts despite high levels of fluorescence interference.     

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.     

The FadL family: unusual transporters for unusual substrates

The FadL family of proteins is responsible for the transport of hydrophobic compounds across the bacterial outer membrane. Two crystal structures of FadL, the long-chain fatty acid transporter from Escherichia coli, were recently determined, showing a novel fold characterized by the combination of a 14-stranded beta barrel and a "hatch" domain that plugs the barrel. Both crystal forms have several bound detergent molecules in the interior of the protein. This, together with differences between the N-terminal conformations of the FadL structures, has led to the proposal of a transport model that is distinct from those of all other known outer membrane transporters. According to this model, the transport of hydrophobic substrates across the outer membrane, as mediated by FadL family members, is based on diffusion, coupled to spontaneous conformational changes in the hatch domain.     

A family of bacteriocin ABC transporters carry out proteolytic processing of their substrates concomitant with export

Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so- called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues In positions -1 and 2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.     

Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates

Two assays for the plasminogen activator urokinase using peptide fluorogenic substrates are described. N-carbobenzoxy-glycyl-glycyl-l-arginine-4-methoxy-β-naphthylamide (CBZ-Gly-Gly-Arg-4MβNA) can be used in a direct assay that is simple, rapid, and sensitive to as little as 0.5 IU/ml urokinase. Additional sensitivity, to 0.01 IU/ml urokinase, is obtained in a second method that uses plamsinogen as the primary substrate followed by a fluorogenic substrate assay employing N-carbobenzoxy-l-alanyl-l-alanyl-l-lysine-4-methoxy-β-naphthylamide (CBZ-Ala-Ala-Lys-4MβNA) as a specific substrate for the activated plasmin. These assays are as sensitive as the best assays presently in use and are simpler to perform. In addition, these assays can readily be used for kinetic analysis of the hydrolytic activity of urokinase or other plasminogen activators.     

Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport

Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P₄-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P₄-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P₄-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Natural history of ABC systems: not only transporters

In recent years, our understanding of the functioning of ABC (ATP-binding cassette) systems has been boosted by the combination of biochemical and structural approaches. However, the origin and the distribution of ABC proteins among living organisms are difficult to understand in a phylogenetic perspective, because it is hard to discriminate orthology and paralogy, due to the existence of horizontal gene transfer. In this chapter, I present an update of the classification of ABC systems and discuss a hypothetical scenario of their evolution. The hypothetical presence of ABC ATPases in the last common ancestor of modern organisms is discussed, as well as the additional possibility that ABC systems might have been transmitted to eukaryotes, after the two endosymbiosis events that led to the constitution of eukaryotic organelles. I update the functional information of selected ABC systems and introduce new families of ABC proteins that have been included recently into this vast superfamily, thanks to the availability of high-resolution three-dimensional structures.