scholarly journals Cloning and functional expression of a cDNA from rat jejunal epithelium encoding a protein (4F2hc) with system y+L amino acid transport activity

1998 ◽  
Vol 330 (2) ◽  
pp. 745-752 ◽  
Author(s):  
Y. M. Sylvia YAO ◽  
R. William MUZYKA ◽  
F. John ELLIOTT ◽  
I. Christopher CHEESEMAN ◽  
D. James YOUNG
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Membrane Transport ◽  
Amino Acid Transport ◽  
Functional Expression ◽  
Amino Acid Transporter ◽  
Transmembrane Domains ◽  
Acid Transport ◽  
Transport Activity ◽  
Cationic Amino Acids

Two different protein families, designated CAT (cationic amino acid transporter) and BAT (broad-specificity amino acid transporter) mediate the plasma membrane transport of cationic amino acids in animal cells. CAT transporters have 12-14 transmembrane domains and are selective for cationic amino acids. BAT proteins, in contrast, have one to four transmembrane domains and induce the transport of both cationic and zwitterionic amino acids when expressed in Xenopus oocytes. Mutations in the human BAT gene cause type I cystinuria, a disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. We have used functional expression cloning in oocytes to isolate a BAT-related cDNA from rat jejunal epithelium. The cDNA encodes the rat 4F2 heavy chain (4F2hc) cell-surface antigen, a 527-residue (60 kDa) protein that is 26% identical in amino acid sequence with rat renal BAT (also known as NBAT/D2). Expression of rat jejunal 4F2hc in oocytes induced the lysine-inhibitable Na+-dependent influx of leucine and the leucine-inhibitable Na+-independent influx of lysine. Lysine efflux was stimulated by extracellular (Na+ plus leucine). These characteristics identify the expressed amino acid transport activity as system y+L, a transporter that has been implicated in basal membrane transport of cationic amino acids in intestine, kidney and placenta.

scholarly journals Amino Acid Transport into Cultured McCoy Cells Infected with Chlamydia trachomatis

2000 ◽  
Vol 68 (9) ◽  
pp. 5439-5442 ◽  
Author(s):  
Angela Harper ◽  
Christopher I. Pogson ◽  
John H. Pearce
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chlamydia Trachomatis ◽  
Amino Acid Transport ◽  
Equilibrium Concentration ◽  
Transport Systems ◽  
Acid Transport ◽  
Entry Rate ◽  
Great Capacity ◽  
Cationic Amino Acids

ABSTRACT Amino acid transport into McCoy cells infected with strains representative of the two major biovars of Chlamydia trachomatis has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms.

scholarly journals Amino acid sensing by enteroendocrine STC-1 cells: role of the Na+-coupled neutral amino acid transporter 2

2010 ◽  
Vol 298 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
Steven H. Young ◽  
Osvaldo Rey ◽  
Catia Sternini ◽  
Enrique Rozengurt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Isobutyric Acid ◽  
Amino Acid Transporter ◽  
Membrane Depolarization ◽  
Neutral Amino Acid ◽  
Acid Transport ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

Evidence that the transport-related proteins BAT and 4F2hc are not specific for amino acids: induction of Na+-dependent uridine and pyruvate transport activity by recombinant BAT and 4F2hc expressed in Xenopus oocytes

1998 ◽  
Vol 76 (5) ◽  
pp. 859-865 ◽  
Author(s):  
Sylvia YM Yao ◽  
William R Muzyka ◽  
Carol E Cass ◽  
Christopher I Cheeseman ◽  
James D Young
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Xenopus Oocytes ◽  
Transport Systems ◽  
Choline Uptake ◽  
Type I ◽  
Organic Substrates ◽  
Acid Transport ◽  
Cationic Amino Acids

Members of the BAT and 4F2hc gene family have one or, in the case of BAT, up to four transmembane domains and induce amino acid transport systems bo,+ (BAT) and y+L (4F2hc) when expressed in Xenopus oocytes. System bo,+ is a Na+-independent process with a broad tolerance for cationic and zwitterionic amino acids, whereas y+L exhibits Na+-independent transport of cationic amino acids (e.g., lysine) and Na+-dependent transport of zwitterionic amino acids (e.g., leucine). Mutations in the human BAT gene are associated with type I cystinuria, a genetic disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. An unresolved question is whether BAT and 4F2hc themselves have catalytic (i.e., transporting) activity or whether they operate as activators of other, as yet unidentified, transporter proteins. In this report, we have investigated the transport of representatives of four different classes of organic substrates in Xenopus oocytes following injection with rat BAT or 4F2hc RNA transcripts: leucine (a control amino acid substrate), uridine (a nucleoside), pyruvate (a monocarboxylate), and choline (an amine). Both recombinant proteins induced small, statistically significant Na+-dependent fluxes of uridine and pyruvate but had no effect on choline uptake. In contrast, control oocytes injected with transcripts for conventional nucleoside and cationic amino acid transporters (rat CNT1 and murine CAT1, respectively) showed no induction of transport of either leucine or pyruvate (CNT1) or uridine or pyruvate (CAT1). These findings support the idea that BAT and 4F2hc are transport activators and minimize the possibility that they have intrinsic transport capability. The transport-regulating functions of these proteins may extend to permeants other than amino acids.Key words: amino acid transport, uridine, pyruvate, BAT, 4F2hc, Xenopus oocytes.

scholarly journals Mutations of the basic amino acid transporter gene associated with cystinuria

1995 ◽  
Vol 310 (3) ◽  
pp. 951-955 ◽  
Author(s):  
K Miyamoto ◽  
K Katai ◽  
S Tatsumi ◽  
K Sone ◽  
H Segawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Transport ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Regulatory Subunit ◽  
Acid Transport ◽  
Transport Activity ◽  
Wild Type ◽  
Neutral Amino Acid Transporter ◽  
Acid Transporter

To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, we analysed the rBAT gene in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator or regulatory subunit by homo- or hetero-multimer complex formation.

Characterization of a mouse colonic system B0+ amino acid transporter related to amino acid absorption in colon

2001 ◽  
Vol 281 (2) ◽  
pp. G365-G370 ◽  
Author(s):  
Shinya Ugawa ◽  
Yoko Sunouchi ◽  
Takashi Ueda ◽  
Eri Takahashi ◽  
Yoshitsugu Saishin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Transport System ◽  
Amino Acid Transport ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Amino Acid Transport System ◽  
Acid Transporter

Previous experiments have shown that an amino acid transport system B0+ transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+ transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+ was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+ mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+ exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+ may absorb amino acids from the intestinal contents in the colon.

scholarly journals Identification of the integrin α3β1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes

1995 ◽  
Vol 311 (3) ◽  
pp. 743-751 ◽  
Author(s):  
J I McCormick ◽  
R M Johnstone
Keyword(s):  
Amino Acid ◽  
Amino Acid Transport ◽  
Plasma Membranes ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Transport Activity ◽  
Western Blots ◽  
Ehrlich Cell ◽  
Cell Plasma ◽  
Beta 1 Integrin

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.

scholarly journals Identification and Functional Characterization of the Lactococcus lactis CodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

2006 ◽  
Vol 188 (9) ◽  
pp. 3280-3289 ◽  
Author(s):  
Chris D. den Hengst ◽  
Maarten Groeneveld ◽  
Oscar P. Kuipers ◽  
Jan Kok
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Lactococcus Lactis ◽  
Amino Acid Transport ◽  
Amino Acid Transporter ◽  
Acid Transport ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
Acid Transporter ◽  
Branched Chain

ABSTRACT Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation. CtrA is required for optimal growth in media containing free amino acids as the only amino acid source. Amino acid transport studies showed that ctrA encodes a secondary amino acid transport system that is specific for branched-chain amino acids (BCAAs) (isoleucine, leucine, and valine) and methionine, which is in disagreement with its previously proposed function (a cationic amino acid transporter), which was assigned based on homology. We propose to rename CtrA BcaP, for branched-chain amino acid permease. BcaP is a member of a group of conserved transport systems, as homologs are widely distributed among gram-positive bacteria. Deletion of bcaP resulted in the loss of most of the BCAA uptake activity of L. lactis, indicating that BcaP is the major BCAA carrier of this organism. Deletion of bcaP together with a second (putative) BCAA permease, encoded by brnQ, further reduced the viability of the strain. DNA microarray analysis showed that deletion of bcaP predominantly affects genes belonging to the regulons of the transcriptional regulator CodY, which is involved in global nitrogen metabolism and needs BCAAs for its activation, and of CmbR, which is involved in sulfur amino acid metabolism.

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