scholarly journals A GC-MS/Single-Cell Method to Evaluate Membrane Transporter Substrate Specificity and Signaling

2021 ◽  
Vol 8 ◽  
Author(s):  
Stephen J. Fairweather ◽  
Shoko Okada ◽  
Gregory Gauthier-Coles ◽  
Kiran Javed ◽  
Angelika Bröer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Vital Role ◽  
Cellular Growth ◽  
Amino Acid Transporters ◽  
Cationic Amino Acids ◽  
Nutrient Signaling

Amino acid transporters play a vital role in metabolism and nutrient signaling pathways. Typically, transport activity is investigated using single substrates and competing amounts of other amino acids. We used GC-MS and LC-MS for metabolic screening of Xenopus laevis oocytes expressing various human amino acid transporters incubated in complex media to establish their comprehensive substrate profiles. For most transporters, amino acid selectivity matched reported substrate profiles. However, we could not detect substantial accumulation of cationic amino acids by SNAT4 and ATB0,+ in contrast to previous reports. In addition, comparative substrate profiles of two related sodium neutral amino acid transporters known as SNAT1 and SNAT2, revealed the latter as a significant leucine accumulator. As a consequence, SNAT2, but not SNAT1, was shown to be an effective activator of the eukaryotic cellular growth regulator mTORC1. We propose, that metabolomic profiling of membrane transporters in Xenopus laevis oocytes can be used to test their substrate specificity and role in intracellular signaling pathways.

Transporters for Cationic Amino Acids in Animal Cells: Discovery, Structure, and Function

1998 ◽  
Vol 78 (2) ◽  
pp. 487-545 ◽  
Author(s):  
R. DEVÉS ◽  
C. A. R. BOYD
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structure And Function ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Cdna Clones ◽  
Animal Cells ◽  
Cationic Amino Acid ◽  
Cationic Amino Acids ◽  
And Function

Devés, R., and C. A. R. Boyd. Transporters for Cationic Amino Acids in Animal Cells: Discovery, Structure, and Function. Physiol. Rev. 78: 487–545, 1998. — The structure and function of the four cationic amino acid transporters identified in animal cells are discussed. The systems differ in specificity, cation dependence, and physiological role. One of them, system y+, is selective for cationic amino acids, whereas the others (B0,+, b0,+, and y+L) also accept neutral amino acids. In recent years, cDNA clones related to these activities have been isolated. Thus two families of proteins have been identified: 1) CAT or cationic amino acid transporters and 2) BAT or broad-scope transport proteins. In the CAT family, three genes encode for four different isoforms [CAT-1, CAT-2A, CAT-2(B) and CAT-3]; these are ∼70-kDa proteins with multiple transmembrane segments ( 12 – 14 ), and despite their structural similarity, they differ in tissue distribution, kinetics, and regulatory properties. System y+is the expression of the activity of CAT transporters. The BAT family includes two isoforms (rBAT and 4F2hc); these are 59- to 78-kDa proteins with one to four membrane-spanning segments, and it has been proposed that these proteins act as transport regulators. The expression of rBAT and 4F2hc induces system b0,+and system y+L activity in Xenopus laevis oocytes, respectively. The roles of these transporters in nutrition, endocrinology, nitric oxide biology, and immunology, as well as in the genetic diseases cystinuria and lysinuric protein intolerance, are reviewed. Experimental strategies, which can be used in the kinetic characterization of coexpressed transporters, are also discussed.

scholarly journals Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

2021 ◽  
Author(s):  
Stephen J Fairweather ◽  
Esther Rajendran ◽  
Martin Blume ◽  
Kiran Javed ◽  
Birte Steinhoefel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Large Family ◽  
Essential Amino Acids ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid ◽  
Intracellular Parasites ◽  
Cationic Amino Acids ◽  
High Concentrations

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is "trans-stimulated" by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

scholarly journals Transcriptome Analysis and Expression of Selected Cationic Amino Acid Transporters in the Liver of Broiler Chicken Fed Diets with Varying Concentrations of Lysine

2020 ◽  
Vol 21 (16) ◽  
pp. 5594
Author(s):  
Collins N. Khwatenge ◽  
Boniface M. Kimathi ◽  
Samuel N. Nahashon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Broiler Chickens ◽  
Molecular Mechanisms ◽  
Cellular Growth ◽  
Control Group ◽  
Amino Acid Transporters ◽  
Potential Candidate ◽  
Cationic Amino Acid ◽  
High Lysine

Amino acids are known to play a key role in gene expression regulation. Amino acid signaling is mediated via two pathways: the mammalian target of rapamycin complex 1 (mTORC1) and the amino acid responsive (AAR) pathways. Cationic amino acid transporters (CATs) are crucial in these pathways due to their sensing, signaling and transport functions. The availability of certain amino acids plays a key role in the intake of other amino acids, hence affecting growth in young birds. However, the specific mechanism for regulating lysine transport for growth is not clear. In this study, we analyze the transcriptome profiles and mRNA expression of selected cationic amino acid transporters in the livers of broilers fed low and high lysine diets. Birds consumed high-lysine (1.42% lysine) or low-lysine (0.85% lysine) diets while the control group consumed 1.14% lysine diet. These concentrations of lysine represent 125% (high lysine), 75% (low lysine) and 100% (control), respectively, of the National Research Council’s (NRC) recommendation for broiler chickens. After comparing the two groups, 210 differentially expressed genes (DEGs) were identified (fold change >1 and false discovery rate (FDR) <0.05). When comparing the high lysine and the low lysine treatments, there were 67 upregulated genes and 143 downregulated genes among these DEGs. Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Gene Ontology (GO) enrichment analysis show that cellular growth, lipid metabolism and lysine metabolism pathways were among the significantly enriched pathways. This study contributes to a better understanding of the potential molecular mechanisms underlying the correlation between lysine intake, body weight gain (BWG) and feed intake (FI) in broiler chickens. Moreover, the DEGs obtained in this study may be used as potential candidate genes for further investigation of broiler growth customized responses to individualized nutrients such as amino acids.

scholarly journals Cryo-EM structure of the human heteromeric amino acid transporter b0,+AT-rBAT

2020 ◽  
Vol 6 (16) ◽  
pp. eaay6379 ◽  
Author(s):  
Renhong Yan ◽  
Yaning Li ◽  
Yi Shi ◽  
Jiayao Zhou ◽  
Jianlin Lei ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Membrane Trafficking ◽  
Binding Pocket ◽  
Disulfide Bridge ◽  
Ligand Molecule ◽  
Amino Acid Transporters ◽  
Cationic Amino Acids

Heteromeric amino acid transporters (HATs) catalyze the transmembrane movement of amino acids, comprising two subunits, a heavy chain and a light chain, linked by a disulfide bridge. The b0,+AT (SLC7A9) is a representative light chain of HATs, forming heterodimer with rBAT, a heavy chain which mediates the membrane trafficking of b0,+AT. The b0,+AT-rBAT complex is an obligatory exchanger, which mediates the influx of cystine and cationic amino acids and the efflux of neutral amino acids in kidney and small intestine. Here, we report the cryo-EM structure of the human b0,+AT-rBAT complex alone and in complex with arginine substrate at resolution of 2.7 and 2.3 Å, respectively. The overall structure of b0,+AT-rBAT exists as a dimer of heterodimer consistent with the previous study. A ligand molecule is bound to the substrate binding pocket, near which an occluded pocket is identified, to which we found that it is important for substrate transport.

scholarly journals Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii

2021 ◽  
Vol 17 (8) ◽  
pp. e1009835
Author(s):  
Stephen J. Fairweather ◽  
Esther Rajendran ◽  
Martin Blume ◽  
Kiran Javed ◽  
Birte Steinhöfel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Large Family ◽  
Essential Amino Acids ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid ◽  
Intracellular Parasites ◽  
Cationic Amino Acids ◽  
High Concentrations

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is ‘trans-stimulated’ by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Placental amino acid transport

1995 ◽  
Vol 268 (6) ◽  
pp. C1321-C1331 ◽  
Author(s):  
A. J. Moe
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Placenta ◽  
Amino Acid Transport ◽  
Single Layer ◽  
Maternal Blood ◽  
Transport Systems ◽  
Amino Acid Transporters ◽  
Acid Transport ◽  
Principal Mechanism

Normal fetal growth and development depend on a continuous supply of amino acids from the mother to the fetus. The placenta is responsible for the transfer of amino acids between the two circulations. The human placenta is hemomonochorial, meaning that the maternal and fetal circulations are separated by a single layer of polarized epithelium called the syncytiotrophoblast, which is in direct contact with maternal blood. Transport proteins located in the microvillous and basal membranes of the syncytiotrophoblast are the principal mechanism for transfer from maternal blood to fetal blood. Knowledge of the function and regulation of syncytiotrophoblast amino acid transporters is of great importance in understanding the mechanism of placental transport and potentially improving fetal and newborn outcomes. The development of methods for the isolation of microvillous and basal membrane vesicles from human placenta over the past two decades has contributed greatly to this understanding. Now a primary cultured trophoblast model is available to study amino acid transport and regulation as the cells differentiate. The types of amino acid transporters and their distribution between the syncytiotrophoblast microvillous and basal membranes are somewhat unique compared with other polarized epithelia. These differences may reflect the unusual circumstance of this epithelium that is exposed to blood on both sides. The current state of knowledge as to the types of transport systems present in syncytiotrophoblast, their regulation, and the effects of maternal consumption of drugs on transport are discussed.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

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