植物PDR型ABC转运蛋白的结构及功能

ATP-结合盒(ATP-binding cassette,ABC)转运蛋白是目前已知最大、功能最广泛的蛋白质家族。多向耐药性(pleiotropic drug resistance,PDR)蛋白是该家族中仅存于植物和真菌中的一个亚族,结构域与其他亚族相反,即核苷酸结合域(nucleotide-binding domain,NBD)位于跨膜结构域(trans-membrane domain,TMD)的N端。目前已发现PDR型转运蛋白具有转运次生代谢产物和参与胁迫反应等方面的功能。植物PDR基因分为5个亚族:I族基因涉及多种生物和非生物胁迫反应,II￣V族基因功能研究甚少。植物PDR基因在器官水平、化学及环境因素影响下具有特异性较好的表达谱。本文系统阐述了植物PDR型转运蛋白基因的进化、结构及其功能,为理解植物PDR型转运蛋白在生物分子转运和复杂生理功能方面提供一个基础框架。     

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.     

The ABC transporters of Saccharomyces cerevisiae

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.     

Peroxisomal ABC transporters

Peroxisomes perform a range of different functions, dependent upon organism, tissue type, developmental stage or environmental conditions, many of which are connected with lipid metabolism. This review summarises recent research on ATP binding cassette (ABC) transporters of the peroxisomal membrane (ABC subfamily D) and their roles in plants, fungi and animals. Analysis of mutants has revealed that peroxisomal ABC transporters play key roles in specific metabolic and developmental functions in different organisms. A common function is import of substrates for beta-oxidation but much remains to be determined concerning transport substrates and mechanisms which appear to differ significantly between phyla.     

Plant ABC transporters

The ATP binding cassette (ABC) superfamily is a large, ubiquitous and diverse group of proteins, most of which mediate transport across biological membranes. ABC transporters have been shown to function not only as ATP-dependent pumps, but also as ion channels and channel regulators. Whilst members of this gene family have been extensively characterised in mammalian and microbial systems, the study of plant ABC transporters is a relatively new field of investigation. Sequences of over 20 plant ABC proteins have been published and include homologues of P-glycoprotein, MRP, PDR5 and organellar transporters. At present, functions have been assigned to a small proportion of these genes and only the MRP subclass has been extensively characterised. This review aims to summarise literature relevant to the study of plant ABC transporters, to review methods of cloning, to discuss the utility of yeast and mammalian systems as models and to speculate on possible roles of uncharacterised ABC transporters in plants.     

Structure of ABC transporters

ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.     

Thermodynamics of ABC transporters

ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion.We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.     

The ATP switch model for ABC transporters

ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.     

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

Structural diversity of ABC transporters

ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity.ATP-binding cassette (ABC) transporters are a large group of membrane protein complexes that couple transport of a substrate across the membrane to the hydrolysis of the phosphate bond between the γ- and the β-phosphate of ATP (Ames et al., 1990; Higgins, 1992; Davidson et al., 2008; Rees et al., 2009). The free energy released when ATP is converted into ADP and orthophosphate (P_i; approximately −50 kJ mol⁻¹ in many cells) can be used to accumulate the transported substrates in, or to remove them from, cellular compartments.In prokaryotes, ABC transporters are localized to the plasma membrane, and ATP is hydrolyzed on the cytoplasmic side. In eukaryotes, ABC transporters are also found in organellar membranes. ATP hydrolysis by organellar ABC transporters takes place on the cytosolic side of the membrane, except for transporters from mitochondria and chloroplasts where the ATP-binding domains of the transporters are located on the matrix or stroma side. The side of the membrane where ATP is bound and hydrolyzed is termed the cis-side, and the opposite side is called the trans-side.ABC transporters can be classified as exporters or importers. Exporters move substrates from the cis-side to the trans-side of the membrane, from the hydrophobic core of the lipid bilayer to the trans-side, or transfer substrates between the inner and outer leaflets of the bilayer. In contrast, importers move substrates from the trans-side to the cis-side. There are a few ABC transporters that do not have a bona fide transport function. Notable examples include the CFTR, which is a gated chloride channel (Aleksandrov et al., 2007), and the sulfonylurea receptor SUR1, which is a regulatory complex associated with a potassium channel (Bryan et al., 2007). There has been tremendous interest in ABC transporters not only from a mechanistic point of view but also because malfunctioning of human ABC transporters leads to a plethora of diseases (see for instance, Silverton et al., 2011), and some ABC exporters are involved in the drug resistance of bacteria and cancer cells (Ambudkar et al., 2003; Davidson et al., 2008).Many excellent reviews on ABC transporters have been published over the past few years and cover the history, structure, mechanism, physiology, and pharmacology of these proteins (Davidson et al., 2008; Rees et al., 2009; Parcej and Tampé, 2010; Eitinger et al., 2011; George and Jones, 2012; Lewis et al., 2012). Because a wealth of new crystal structures has been determined lately, here we provide an update on the structural diversity of ABC transporters.

Overview

Similar to other membrane transport proteins, ABC transporters adopt at least two conformations in which the substrate-binding site is accessible from either the cis-side or the trans-side. Alternation between the two conformations allows substrate translocation across the membrane (“alternating access” model; Jardetzky, 1966; Tanford, 1982). The binding of substrate on one side of the membrane and release on the opposite side are coordinated by ATP binding and hydrolysis, and ADP and P_i release. Several ABC transporters have been crystallized in different conformations (see

The ABC transporters in lipid flux and atherosclerosis

Atherosclerotic cardiovascular disease is the leading cause of morbidity and mortality in the United States and in many other countries. Dysfunctional lipid homeostasis plays a central role in the initiation and progression of atherosclerotic lesions. The ATP-binding cassette (ABC) transporters are transmembrane proteins that hydrolyze ATP and use the energy to drive the transport of various molecules across cell membranes. Several ABC transporters play a pivotal role in lipid trafficking. They are critically involved in cholesterol and phospholipid efflux and reverse cholesterol transport (RCT), processes that maintain cellular cholesterol homeostasis and protect arteries from atherosclerosis. In this article we provide a review of the current literature on the biogenesis of ABC transporters and highlight their proposed functions in atheroprotection.     

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.     

A fourth gene from the Candida albicans CDR family of ABC transporters

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I.1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.     

The controversial role of ABC transporters in clinical oncology

The phenomenon of multidrug resistance in cancer is often associated with the overexpression of the ABC (ATP-binding cassette) transporters Pgp (P-glycoprotein) (ABCB1), MRP1 (multidrug resistance-associated protein 1) (ABCC1) and ABCG2 [BCRP (breast cancer resistance protein)]. Since the discovery of Pgp over 35 years ago, studies have convincingly linked ABC transporter expression to poor outcome in several cancer types, leading to the development of transporter inhibitors. Three generations of inhibitors later, we are still no closer to validating the 'Pgp hypothesis', the idea that increased chemotherapy efficacy can be achieved by inhibition of transporter-mediated efflux. In this chapter, we highlight the difficulties and past failures encountered in the development of clinical inhibitors of ABC transporters. We discuss the challenges that remain in our effort to exploit decades of work on ABC transporters in oncology. In learning from past mistakes, it is hoped that ABC transporters can be developed as targets for clinical intervention.     

The role of ABC transporters in cuticular lipid secretion

The aerial surfaces of plants are enveloped by a waxy cuticle, which among other functions serves as a barrier to limit non-stomatal water loss and defend against pathogens. The cuticle is a complex three-dimensional structure composed of cutin (a lipid polyester matrix) and waxes (very long chain fatty acid derivatives), which are embedded within and layered on top of the cutin matrix. Biosynthesis of cuticular lipids is believed to take place solely within aerial epidermal cells. Once synthesized, both the waxes and the cutin precursors must leave the cytoplasm, pass through the hydrophilic apoplastic space, and finally assemble to form the cuticle. These processes of secretion and assembly are essentially unknown. Initial steps toward our understanding of these processes were the characterization of CER5/ABCG12/WBC12 and more recently ABCG11/WBC11, a pair of ABC transporters required for cuticular lipid secretion. ABCG12 is involved in wax secretion, as mutations in this gene result in a lower surface-load of wax and a concomitant accumulation of lipidic inclusions within the epidermal cell cytoplasm. Mutations in ABCG11 result in a similar wax phenotype as cer5 and similar cytoplasmic inclusions. In contrast to cer5, however, abcg11 mutants also show significantly reduced cutin, post-genital organ fusions, and reduced growth and fertility. Thus, for the first time, a transporter is implicated in cutin accumulation. This review will discuss the secretion of cuticular lipids, focusing on ABCG12, ABCG11 and the potential involvement of other ABC transporters in the ABCG subfamily.