Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L. A candidate gene for lysinuric protein intolerance

We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively. y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e. 4F2hc) and approximately 40 kDa (y+LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity. These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y+LAT-1 mRNA is expressed in kidney > peripheral blood leukocytes > lung > placenta = spleen > small intestine. The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P. , Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479-1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI.     

N-system amino acid transport at the blood--CSF barrier

Despite L-glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in L-[14C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na(+)-dependent mechanism. The Na(+)-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na(+)-dependent uptake was inhibited neither by the A-system substrate alpha-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and L-glutamate gamma-hydroxamate, three N-system substrates. Replacement of Na+ with Li+ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The Vmax and Km for glutamine transport by this system were 8.1 +/- 0.3 nmol/mg/min and 3.3 +/- 0.4 mM, respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Renal amino acid transport in adults with oxidative phosphorylation diseases

The clinical manifestations of mitochondrial DNA (mtDNA) mutations depend on a variety of factors including ratios of normal to abnormal mtDNA and tissue-specific differences in ATP production by oxidative phosphorylation (OXPHOS). In order to investigate the effects of OXPHOS defects on renal tubule function, we characterized sodium-coupled transport processes in six individuals with OXPHOS diseases. Pathogenic mtDNA mutations were identified in five of these individuals. Sodium coupled transport processes were evaluated by determining fractional excretions of amino acids, glucose, lactate, urate, and phosphate in patients and controls. Four of the six individuals had high fractional excretions of neutral amino acids, indicating abnormal renal tubule reabsorbtion of these amino acids. Abnormalities in fractional excretions of lactate, glucose, urate, and phosphate were less pronounced. These results demonstrate that sodium-coupled transport processes in the kidney are sensitive to OXPHOS impairment. When abnormalities in these processes are encountered, an OXPHOS disease should be included in the differential diagnosis.     

Assigning amino acid sequences to 3-dimensional protein folds

With the advent of genome sequencing projects, the amino acid sequences of thousands of proteins are determined every year. Each of these protein sequences must be identified with its function and its 3-dimensional structure for us to gain a full understanding of the molecular biology of organisms. To meet this challenge, new methods are being developed for fold recognition, the computational assignment of newly determined amino acid sequences to 3-dimensional protein structures. These methods start with a library of known 3-dimensional target protein structures. The new probe sequence is then aligned to each target protein structure in the library and the compatibility of the sequence for that structure is scored. If a target structure is found to have a significantly high compatibility score, it is assumed that the probe sequence folds in much the same way as the target structure. The fundamental assumptions of this approach are that many different sequences fold in similar ways and there is a relatively high probability that a new sequence possesses a previously observed fold. We review various approaches to fold recognition and break down the process into its main steps: creation of a library of target folds; representation of the folds; alignment of the probe sequence to a target fold using a sequence-to-structure compatibility scoring function; and assessment of significance of compatibility. We emphasize that even though this new field of fold recognition has made rapid progress, technical problems remain to be solved in most of the steps. Standard benchmarks may help identify the problem steps and find solutions to the problems.     

Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4^{+ channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.}     

Identification of a membrane protein involved in mitochondrial phosphate transport

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two-SH-groups of the transport system with low concentrations of mersalyl. Subsequently, the other free-SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent inbucation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to one-SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjected to dodecylsulfate gel electrophoresis. The followin results were obtained. 1. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H-A1-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 degrees C and at 0 degrees C. 2. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 +/- 800. 3. The amount of one of the two-SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.     

True digestibility of amino acids and protein in pigs with 13C as a label to determine endogenous amino acid excretion

The objective of this study was to determine whether differential labeling of 13C occurs in pigs fed diets with different 13C abundances and, if so, to use 13C as a label to determine true amino acid digestibility. Forty-eight pigs averaging 10.5 kg BW were fed dietary treatments consisting of a corn-corn gluten meal-crystalline amino acid diet (C-CGM) and a wheat-soybean meal diet (W-SBM). The 13C abundance of the amino acid fraction (AAF) of the C-CGM and W-SBM diets averaged delta 13C -14.19 and -26.36/1000, respectively. Three pigs/treatment group were killed when groups averaged 10.5 (initial), 22.9, and 46.6 kg BW, and AAF of organs were analyzed for 13C abundance. Carbon 13 in empty body AAF increased (-18.14, -13.98, and -12.66/1000) with increasing body weight in pigs fed the C-CGM diet and decreased (-18.06, -22.78, and -24.76/1000) in pigs fed the W-SBM diet. Liver, small intestine, and longissimus muscle tissues showed similar trends. Each tissue had dietary treatment effects (P < .001) and dietary treatment x weight group (P < .001) interactions. Ten pigs averaging 55.0 kg BW from each treatment group were assigned to metabolism cages and fed at 0700 and 1900. Six of these pigs from each treatment group were implanted with T-cannulas in the ileum and given a 17-d recovery period. At 1900 on d 0 of the collection phase, pigs were switched to the opposite diet that contained chromic oxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insertion of a bacterial secondary transport protein in the endoplasmic reticulum membrane

The sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) contains 12 hydrophobic potential transmembrane domains. Surprisingly, an alkaline phosphatase fusion study in Escherichia coli has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). To provide independent data on the topology and mode of membrane insertion of CitS, we have investigated its insertion into the endoplasmic reticulum (ER) membrane. By using in vitro translation of model proteins in the presence of dog pancreas microsomes, each of the putative transmembrane segments of CitS was assayed for its potency to insert into the ER membrane, both as an isolated segment as well as in the context of COOH-terminal truncation mutants. All 12 segments were able to insert into the membrane as Ncyt-Clum signal anchor sequences. In a series of COOH-terminal truncation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segments VIII and IX, which, according to the alkaline phosphatase fusion study, are in the periplasm of E. coli, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segments and also demonstrate that the sequence requirements for signal anchor and stop transfer function are different.     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b.wt. per hour), or alanine (90 mg/100 g b.wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8 micrograms/100 g b.wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 +/- 0.10 vs. 0.62 +/- 0.07 ml/min x 100 g b.wt.) and a distinct increase in sodium excretion (2.98 +/- 0.55 vs. 4.97 +/- 0.71 muval/100 g b.wt. x 20 min). After loading with p-aminohippurate (PAH; 200 mg/100 g b.wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 +/- 0.04 vs. EGF: 0.18 +/- 0.03 mg/100 g b.wt. x 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 +/- 18 vs. 775 +/- 32 mg/100 g b.wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or triiodothyronine, its effect is only of a moderate degree.     

A method for identifying the carboxy terminal amino acid of a protein

A highly sensitive new method for identifying the carboxy terminus of a protein was developed. The carboxyl terminal amino acid was racemized by reaction with acetic anhydride. The resulting modified protein was subjected to acid hydrolysis. The hydrolysate was derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate to give fluorescent amino acid diastereomers. The amino acid diastereomers were separated on a reversed-phase column. Only carboxyl terminal amino acids give a D-amino acid. Application of this method was described for the isolation and identification of carboxyl terminal peptides from an enzymatic digest of a protein.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The effects of systemic administration of muscle extract on normal muscle were studied. Male Wistar rats received intraperitoneal injections of normal and denervated muscle extract over 5 consecutive days. Soleus muscles were then submitted to histological, histochemical and quantitative-morphometric analysis. The group receiving denervated muscle extract showed considerable muscle fiber hypertrophy, together with the formation of new fibers suggestive of hyperplasia. The systemic administration of denervated muscle extract was shown to have a considerable myotrophic effect on normal muscle, evident in the hypertrophy and hyperplasia of muscle fibers.     

Antibody to tumor necrosis factor attenuates endotoxin-stimulated amino acid transport in rat liver

BACKGROUND: Endotoxemia stimulates amino acid consumption by the liver, but the regulation of this response is poorly understood. We studied the effect of Escherichia coli endotoxin (lipopolysaccharide) on hepatic carrier-mediated plasma membrane amino acid transport and the role of the cytokine tumor necrosis factor-alpha (TNF) in regulating this transport activity. METHODS: We investigated the activities of the Na(+)-dependent amino acid transport systems A, ASC, and N in hepatic plasma membrane vesicles prepared from rats treated with endotoxin in vivo. Vesicle purity and functionality were evaluated by assaying marker enzymes and by the presence of classic overshoots. RESULTS: Endotoxin treatment did not alter sodium transport but resulted in time- and dose-dependent 6-fold (system A), 3.5-fold (system N), and 3-fold (system ASC) increases in transport activity secondary to an increase in carrier maximum velocity. Lipopolysaccharide treatment did not alter transporter affinity or plasma membrane sodium transport. Transport activity increased within 2 hours of endotoxin administration, peaked at 4 hours after exposure to lipopolysaccharide, and returned to basal levels within 24 hours. Pretreatment of animals with an anti-TNF monoclonal antibody diminished the endotoxin-induced enhancement in transport activity by 50% to 75% by decreasing carrier maximum velocity. In contrast, when the antibody was given after endotoxin challenge, transport activity was not attenuated. CONCLUSIONS: The marked acceleration in hepatic amino acid uptake that occurs during endotoxemia is secondary to an increased Na(+)-dependent hepatocyte plasma membrane transport activity and is mediated, in large part, by the cytokine TNF.     

Intracellular protein transport to the thyrocyte plasma membrane: potential implications for thyroid physiology

Calculations of stopping power ratios, water to air, for the determination of absorbed dose to water in clinical proton beams using ionization chamber measurements have been undertaken using the Monte Carlo method. A computer code to simulate the transport of protons in water (PETRA) has been used to calculate sw.air-data under different degrees of complexity, ranging from values based on primary protons only to data including secondary electrons and high-energy secondary protons produced in nonelastic nuclear collisions. All numerical data are based on ICRU 49 proton stopping powers. Calculations using primary protons have been compared to the simple continuous slowing-down approximation (c.s.d.a.) analytical technique used in proton dosimetry protocols, not finding significant differences that justify elaborate Monte Carlo simulations except beyond the mean range of the protons (the far side of the Bragg peak). The influence of nuclear nonelastic processes, through the detailed generation and transport of secondary protons, on the calculated stopping-power ratios has been found to be negligible. The effect of alpha particles has also been analysed, finding differences smaller than 0.1% from the results excluding them. Discrepancies of up to 0.6% in the plateau region have been found, however, when the production and transport of secondary electrons are taken into account. The large influence of nonelastic nuclear interactions on proton depth-dose distributions shows that the removal of primary protons from the incident beam decreases the peak-to-plateau ratio by a large factor, up to 40% at 250 MeV. It is therefore emphasized that nonelastic nuclear reactions should be included in Monte Carlo simulations of proton beam depth-dose distributions.     

Direct binding of the MHC class I molecule H-2Ld to CD8: interaction with the amino terminus of a mature cell surface protein

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.     

The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.     

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/M), X(aromatic/beta-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.     

Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells

System A for neutral amino acid transport is increased by hypertonic shock in NBL-1 cells previously induced to express system A activity by amino acid starvation. The hypertonicity-mediated effect can be blocked by cycloheximide but is insensitive to tunicamycin. The activity induced may be inactivated irreversibly by the addition of system A substrates, by a rapid mechanism insensitive to cycloheximide. In CHO-K1 cells, hypertonicity increases system A activity, as has been shown in NBL-1 cells. This effect is additive to the activity produced by derepression of system A by amino acid starvation and is insensitive to tunicamycin. Furthermore, the alanine-resistant mutant CHO-K1 alar4, which bears a mutation affecting the regulatory gene R1, involved in the derepression of system A activity after amino acid starvation, is still able to respond to the hypertonic shock by increasing system A activity to a level similar to that described in hypertonicity-induced derepressed CHO-K1 (wild type) cells. These results suggest (i) that the hypertonicity-mediated increase of system A activity occurs through a mechanism other than that involved in system A derepression and (ii) that a regulatory protein coded by an osmotically sensitive gene is responsible for further activation of preexisting A carriers.