PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

Growth in normal and tumour cells is regulated by evolutionarily conserved extracellular inputs from the endocrine insulin receptor (InR) signalling pathway and by local nutrients. Both signals modulate activity of the intracellular TOR kinase, with nutrients at least partly acting through changes in intracellular amino acid levels mediated by amino acid transporters. We show that in Drosophila, two molecules related to mammalian proton-assisted SLC36 amino acid transporters (PATs), CG3424 and CG1139, are potent mediators of growth. These transporters genetically interact with TOR and other InR signalling components, indicating that they control growth by directly or indirectly modulating the effects of TOR signalling. A mutation in the CG3424 gene, which we have named pathetic (path), reduces growth in the fly. In a heterologous Xenopus oocyte system, PATH also activates the TOR target S6 kinase in an amino acid-dependent way. However, functional analysis reveals that PATH has an extremely low capacity and an exceptionally high affinity compared with characterised human PATs and the CG1139 transporter. PATH and potentially other PAT-related transporters must therefore control growth via a mechanism that does not require bulk transport of amino acids into the cell. As PATH is likely to be saturated in vivo, we propose that one specialised function of high-affinity PAT-related molecules is to maintain growth as local nutrient levels fluctuate during development.     

A Comparison of the Sucrose Transporter Systems of Different Plant Species

Abstract: The sucrose uptake behaviour of many different plant species is characterised by the presence of at least two components with distinct kinetic properties. These include at least one high-affinity and one low-affinity transport system. All known sucrose transporters from higher plants fall into one of three large subfamilies, according to phylogenetic analysis. Apparently, the largest subfamily, the SUT1 subfamily, exclusively consists of high-affinity sucrose transporters from dicotyledons, whereas none of the transporters from monocotyledonous plants groups within this subfamily. The other two subfamilies of sucrose transporter-like proteins are either low-affinity transporter or putative sucrose-sensing proteins. Most of the known sucrose transporters from monocotyledons are closely related to the SUT2 subfamily and include high-affinity transporters, suggesting a different evolutionary origin of dicotyledonous and monocotyledonous sucrose transporter gene families.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction.

Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl- dependent serotonin uptake, with estimated Km and Vmax values of 436 +/- 19 nm and 3.8 +/- 0.6 x 10-18 mol.cell.min-1, respectively. The Manduca high affinity Na+/Cl- dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 micro m) and cocaine (IC50 = 12.89 micro m). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148-152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 micro m). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1-146/MasSERT106-587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 micro m) compared to the human (IC50 = 0.431 micro m) and Manduca serotonin transporters. The chimera MasSERT1-67/hSERT109-630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 micro m) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.     

SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction

BackgroundAn important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.Methodology/principal findingsWe identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these ‘knockdowns’ reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.Conclusions/significanceOur analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.     

Structure-function analysis of a novel member of the LIV-1 subfamily of zinc transporters, ZIP14

Here, we report the first investigation of a novel member of the LZT (LIV-1 subfamily of ZIP zinc Transporters) subfamily of zinc influx transporters. LZT subfamily sequences all contain a unique and highly conserved metalloprotease motif (HEXPHEXGD) in transmembrane domain V with both histidine residues essential for zinc transport by ZIP (Zrt-, Irt-like Proteins) transporters. We investigate here whether ZIP14 (SLC39A14), lacking the initial histidine in this motif, is still able to transport zinc. We demonstrate that this plasma membrane located glycosylated protein functions as a zinc influx transporter in a temperature-dependant manner.     

Multiple nutrient transporters enable cells to mitigate a rate-affinity tradeoff

Eukaryotic genomes often encode multiple transporters for the same nutrient. For example, budding yeast has 17 hexose transporters (HXTs), all of which potentially transport glucose. Using mathematical modelling, we show that transporters that use either facilitated diffusion or symport can have a rate-affinity tradeoff, where an increase in the maximal rate of transport decreases the transporter’s apparent affinity. These changes affect the import flux non-monotonically, and for a given concentration of extracellular nutrient there is one transporter, characterised by its affinity, that has a higher import flux than any other. Through encoding multiple transporters, cells can therefore mitigate the tradeoff by expressing those transporters with higher affinities in lower concentrations of nutrients. We verify our predictions using fluorescent tagging of seven HXT genes in budding yeast and follow their expression over time in batch culture. Using the known affinities of the corresponding transporters, we show that their regulation in glucose is broadly consistent with a rate-affinity tradeoff: as glucose falls, the levels of the different transporters peak in an order that mostly follows their affinity for glucose. More generally, evolution is constrained by tradeoffs. Our findings indicate that one such tradeoff often occurs in the cellular transport of nutrients.     

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate‐dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.     

A functional-phylogenetic classification system for transmembrane solute transporters.

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.     

Molecular and functional characterization of a family of amino acid transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.     

Plant pleiotropic drug resistance transporters： Transport mechanism,gene expression,and function

Pleiotropic drug resistance （PDR） transporters belonging to the ABCG subfamily of ATP-binding cassette （ABC） transporters are identified only in fungi and plants. Members of this family are expressed in plants in response to various biotic and abiotic stresses and transport a diverse array of moleculesacross membranes, Although their detailed transport mechanism is largely unknown, they play important roles in detoxification processes, preventing water loss, transport of phytohormones, and secondary metabolites. This review provides insights into transport mechanisms of plant PDR transporters, their expression profiles, and multitude functions in plants.     

Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.     

Identification and molecular cloning of a functional GDP-fucose transporter in Drosophila melanogaster

Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter.     

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.     

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.     

Plant pleiotropic drug resistance transporters:Transport mechanism,gene expression,and function

Pleiotropic drug resistance(PDR) transporters belonging to the ABCG subfamily of ATP-binding cassette(ABC)transporters are identified only in fungi and plants. Members of this family are expressed in plants in response to various biotic and abiotic stresses and transport a diverse array of molecules across membranes. Although their detailed transport mechanism is largely unknown, they play important roles in detoxification processes, preventing water loss, transport of phytohormones,and secondary metabolites. This review provides insights into transport mechanisms of plant PDR transporters, their expression profiles, and multitude functions in plants.