Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood-brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1(+)) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two (Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y(+)Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

The SLC3 and SLC7 families of amino acid transporters

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance.

The small SLC43 family: facilitator system l amino acid transporters and the orphan EEG1

The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.

Peptide and amino acid transporters are differentially regulated during seed development and germination in faba bean

Two peptide transporter (PTR) homologs have been isolated from developing seeds of faba bear, (Vicia faba). VfPTR1 was shown to be a functional peptide transporter through complementation of a yeast mutant. Expression patterns of VfPTR1 and VfPTR2 as well as of the amino acid permease VfAAP1 (Miranda et al., 2001) were compared throughout seed development and germination. In developing seeds, the highest levels of VfPTR1 transcripts were reached during midcotyledon development, whereas VfAAP1 transcripts were most abundant during early cotyledon development, before the appearance of storage protein gene transcripts, and were detectable until late cotyledon development. During early germination, VfPTR1 mRNA appeared first in cotyledons and later, during seedling growth, also in axes and roots. Expression of VfPTR2 and VfAAP1 was delayed compared with VfPTR1, and was restricted to the nascent organs of the seedlings. Localization of VfPTR1 transcripts showed that this FTR is temporally and spatially regulated during cotyledon development. In germinating seeds, VfPTR1 mRNA was localized in root hairs and root epidermal cells, suggesting a role in nutrient uptake from the soil. In seedling roots, VfPTR1 was repressed by a dipeptide and by an amino acid, whereas nitrate was without influence.

High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.

Conservation of amino acid transporters in fungi, plants and animals

When comparing the transporters of three completely sequenced eukaryotic genomes - Saccharomyces cerevisiae, Arabidopsis thaliana and Homo sapiens - transporter types can be distinguished according to phylogeny, substrate spectrum, transport mechanism and cell specificity. The known amino acid transporters belong to five different superfamilies. Two preferentially Na+-coupled transporter superfamilies are not represented in them yeast and Arabidopsis genomes, whereas the other three groups, which often function as H+-coupled systems, have members in all investigated genomes. Additional superfamilies exist for organellar transport, including mitochondrial and plastidic carriers. When used in combination with phylogenetic analyses, functional comparison might aid our prediction of physiological functions for related but uncharacterized open reading frames.

Functional and structural characterization of the first prokaryotic member of the L-amino acid transporter (LAT) family: a model for APC transporters

We have identified YkbA from Bacillus subtilis as a novel member of the L-amino acid transporter (LAT) family of amino acid transporters. The protein is approximately 30% identical in amino acid sequence to the light subunits of human heteromeric amino acid transporters. Purified His-tagged YkbA from Escherichia coli membranes reconstituted in proteoliposomes exhibited sodium-independent, obligatory exchange activity for L-serine and L-threonine and also for aromatic amino acids, albeit with less activity. Thus, we propose that YkbA be renamed SteT (Ser/Thr exchanger transporter). Kinetic analysis supports a sequential mechanism of exchange for SteT. Freeze-fracture analysis of purified, functionally active SteT in proteoliposomes, together with blue native polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized purified SteT, suggest that the transporter exists in a monomeric form. Freeze-fracture analysis showed spherical particles with a diameter of 7.4 nm. Transmission electron microscopy revealed elliptical particles (diameters 6 x 7 nm) with a distinct central depression. To our knowledge, this is the first functional characterization of a prokaryotic member of the LAT family and the first structural data on an APC (amino acids, polyamines, and choline for organocations) transporter. SteT represents an excellent model to study the molecular architecture of the light subunits of heteromeric amino acid transporters and other APC transporters.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc.

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.