Emerging new paradigms for ABCG transporters

Every cell is separated from its external environment by a lipid membrane. Survival depends on the regulated and selective transport of nutrients, waste products and regulatory molecules across these membranes, a process that is often mediated by integral membrane proteins. The largest and most diverse of these membrane transport systems is the ATP binding cassette (ABC) family of membrane transport proteins. The ABC family is a large evolutionary conserved family of transmembrane proteins (> 250 members) present in all phyla, from bacteria to Homo sapiens, which require energy in the form of ATP hydrolysis to transport substrates against concentration gradients. In prokaryotes the majority of ABC transporters are involved in the transport of nutrients and other macromolecules into the cell. In eukaryotes, with the exception of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), ABC transporters mobilize substrates from the cytoplasm out of the cell or into specific intracellular organelles. This review focuses on the members of the ABCG subfamily of transporters, which are conserved through evolution in multiple taxa. As discussed below, these proteins participate in multiple cellular homeostatic processes, and functional mutations in some of them have clinical relevance in humans.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Close approximation of putative alpha -helices II, IV, VII, X, and XI in the translocation pathway of the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus belongs to a family of transporters in which putative alpha-helices II and IV have been implicated in cation binding and the coupled transport of the substrate and the cation. Here, the analysis of site-directed mutants shows that a positive and negative charge at positions 64 and 71 in helix II are essential for transport, but not for lactose binding. The conservation of charge/side-chain properties is less critical for Glu-67 and Ile-70 in helix II, and Asp-133 and Lys-139 in helix IV, but these residues are important for the coupled transport of lactose together with a proton. The analysis of second-site suppressor mutants indicates an ion pair exists between helices II and IV, and thus a close approximation of these helices can be made. The second-site suppressor analysis also suggests ion pairing between helix II and the intracellular loops 6-7 and 10-11. Because the C-terminal region of the transmembrane domain, especially helix XI and loop 10-11, is important for substrate binding in this family of proteins, we propose that sugar and proton binding and translocation are performed by the joint action of these regions in the protein. Indeed, substrate protection of maleimide labeling of single cysteine mutants confirms that alpha-helices II and IV are directly interacting or at least conformationally involved in sugar binding and/or translocation. On the basis of new and published data, we reason that the helices II, IV, VII, X, and XI and the intracellular loops 6-7 and 10-11 are in close proximity and form the binding sites and/or the translocation pathway in the transporters of the galactosides-pentosides-hexuronides family.     

高粱SUT基因家族的生物信息学分析

蔗糖转运蛋白(sucrose transporters,SUTs)属于跨膜转运蛋白,大多数参与蔗糖的吸收和转运。迄今为止,对高粱蔗糖转运蛋白知之甚少,为进一步研究高粱蔗糖转运蛋白家族(SbSUTs),本研究利用生物信息学方法对SbSUTs的6个成员(编号SbSUT1~SbSUT6)进行蛋白理化性质、基因结构、蛋白结构、同源性及系统进化树构建等分析。结果表明:SbSUTs是一种无信号肽、定位于质膜和叶绿体类囊膜上的疏水性膜蛋白;SbSUTs均具有GPH结构功能域,是高度保守的蛋白;α-螺旋和无规卷曲是主要的二级结构元件,其三级结构较为相似。本研究为探究SbSUTs蛋白家族在高粱的蔗糖吸收及转运中的功能提供理论依据。     

Phylogenetic relationships within cation transporter families of Arabidopsis

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.     

The SLC6/NSS family members KAAT1 and CAATCH1 have weak chloride dependence

KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta.1,2 They are members of the SLC6/NSS family, which groups membrane proteins that use Na+, K+, and Cl- gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT)3 has contributed significantly to understanding of the structure–function relationship in NSS proteins. Transport by AaLeuT is Cl- independent, whereas many neurotransmitter:sodium symporters like serotonin transporter (SERT), GABA transporter (GAT1), dopamine transporter, and norephinephrine transporter, among others, are strongly Cl- dependent.4 A single Cl- ion is found bound to one of the extracellular loops, EL2 in AaLeuT. The Cl- is 20 Ã… away from the Na and leucine binding sites, and thus it is unclear whether this Cl- binding site is physiologically important. The nature of the association of Cl- ions with these proteins during transport remains to be resolved. The Cl- binding site of two members of the family, the serotonin transporter SERT 4 and the GABA transporter GAT1 5, has been recently modelled on the basis of their functional properties and by structural homology to AaLeuT. The analyses have highlighted the role of a serine residue, that in the Cl--independent AaLeuT corresponds to Glu 290, and of an asparagine (Asn 286) that also contributes to the coordination of Na+ in the Na1 binding site of AaLeuT. KAAT1 and CAATCH1 are able to transport different amino acids depending on the contransported cation (Na+ or K+) but their Cl- dependence is not completely defined yet. With the aim to clarify the role exerted by chloride in SLC6/NSS transporters, the Cl--dependence of KAAT1 and CAATCH1 have been investigated by the expression in Xenopus laevis oocytes and the measurement of induced amino acid uptakes. Despite KAAT1 and CAATCH1 posses the same residue of serine (Ser342, KAAT1 numbering) present in strictly chloride dependent transporters, their transport activities resulted weakly Cl--dependent compared to GAT1. By analysis of the pH dependence of the KAAT1 and CAATCH1 transport activity, we obtained more information to define their (particular) peculiar Cl- dependence.     

Tyrosine residues affecting sodium stimulation of carnitine transport in the OCTN2 carnitine/organic cation transporter

Primary carnitine deficiency is a disorder of fatty acid oxidation caused by mutations in the Na+-dependent carnitine/organic cation transporter OCTN2. Studies with tyrosyl group-modifying reagents support the involvement of tyrosine residues in Na+ binding by sodium-coupled transporters. Here we report two new patients with carnitine deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (Glu-452) previously shown to affect sodium stimulation of carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in Chinese hamster ovary cells, increased the concentration of sodium required to half-maximally stimulate carnitine transport from 14.8 +/- 1.8 to 34.9 +/- 5.8 mM (p<0.05), whereas Y447C completely abolished carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal carnitine transport in Y449F but resulted in markedly impaired carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate carnitine transport to 57.8 +/- 7.4 mM (p<0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium indicating that their effect on carnitine transport was specific and likely associated with the impaired sodium stimulation of carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to carnitine. Confocal microscopy of OCTN2 transporters tagged with green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, whereas Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2.     

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.     

Mutations in novel organic cation transporter (OCTN2), an organic cation/carnitine transporter, with differential effects on the organic cation transport function and the carnitine transport function

Novel organic cation transporter (OCTN2) is an organic cation/carnitine transporter, and two missense mutations, L352R and P478L, in OCTN2 have been identified as the cause for primary carnitine deficiency. In the present study, we assessed the influence of these two mutations on the carnitine transport function and the organic cation transport function of OCTN2. The L352R mutation resulted in a complete loss of both transport functions. In contrast, the P478L mutation resulted in a complete loss of only the carnitine transport function but significantly stimulated the organic cation transport function. Studies with human OCTN2/rat OCTN2 chimeric transporters indicated that the carnitine transport site and the organic cation transport site were not identical. Because carnitine transport is Na(+)-dependent whereas organic cation transport is Na(+)-independent, we investigated the possibility that the P478L mutation affected Na(+) binding. The Na(+) activation kinetics were found to be similar for the P478L mutant and wild type OCTN2. We then mutated nine different tyrosine residues located in or near transmembrane domains and assessed the transport function of these mutants. One of these mutations, Y211F, was found to have differential influence on the two transport activities of OCTN2 as did the P478L mutation. However, the Na(+) activation kinetics were not affected. These findings are of clinical relevance to patients with primary carnitine deficiency because whereas each and every mutation in these patients is expected to result in the loss of the carnitine transport function, all of these mutations may not interfere with the organic cation transport function.     

Potassium transporters in plants--involvement in K+ acquisition, redistribution and homeostasis

Potassium is a major plant nutrient which has to be accumulated in great quantity by roots and distributed throughout the plant and within plant cells. Membrane transport of potassium can be mediated by potassium channels and secondary potassium transporters. Plant potassium transporters are present in three families of membrane proteins: the K(+) uptake permeases (KT/HAK/KUP), the K(+) transporter (Trk/HKT) family and the cation proton antiporters (CPA). This review will discuss the contribution of members of each family to potassium acquisition, redistribution and homeostasis.     

The role of the STAS domain in the function and biogenesis of a sulfate transporter as probed by random mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

Structure and evolution of the plant cation diffusion facilitator family of ion transporters

Background  

Members of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. The spectrum of cations known to be transported by proteins of the CDF family include Zn, Fe, Co, Cd, and Mn. Members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. CDF families range in size from nine members in Selaginella moellendorffii to 19 members in Populus trichocarpa. Phylogenetic analysis suggests that the CDF family has expanded within plants, but a definitive plant CDF family phylogeny has not been constructed.     

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.     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.     

Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris

The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.     

Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter.

The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease.