Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation.

Amino acid deprivation activates the amino acid response (AAR) pathway that enhances transcription of genes containing an amino acid response element (AARE). The present data reveal a quantitative difference in the response to deprivation of individual amino acids. The AAR leads to increased eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and ATF4 translation. When HepG2 cells were deprived of an individual essential amino acid, p-eIF2alpha and activating transcription factor 4 were increased, but the correlation was relatively weak. Complete amino acid starvation in either Earle's balanced salt solution or Krebs-Ringer bicarbonate buffer (KRB) resulted in activation of transcription driven by a SNAT2 genomic fragment that contained an AARE. However, for the KRB, a proportion of the transcription was AARE-independent suggesting that amino acid-independent mechanisms were responsible. Therefore, activation of AARE-driven transcription is triggered by a deficiency in any one of the essential amino acids, but the response is not uniform. Furthermore, caution must be exercised when using a medium completely devoid of amino acids.

Eukaryotic gene expression: metabolite control by amino acids.

Our understanding of the metabolite control in mammalian cells lags far behind that in prokaryotes. This is particularly true for amino-acid-dependent gene expression. Few proteins have been identified for which synthesis is selectively regulated by amino-acid availability, and the mechanisms for control of transcription and translation in response to changes in amino-acid availability have not yet been elucidated. The intimate relationship between amino-acid supply and the fundamental cellular process of protein synthesis makes amino-acid-dependent control of gene expression particularly important. Future studies should provide important insight into amino-acid and other nutrient signaling pathways, and their impact on cellular growth and metabolism.

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles.

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity.

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.

Energization of amino acid transport in energy-depleted Ehrlich cells and plasma membrane vesicles.

We redirect attention to contributions to the energization, of the active transport of amino acids in the Ehrlich cell, beyond the known energization, by down-gradient comigration of Na+, beyond possible direct energization by coupling to ATP breakdown, and beyond known energization by exchange with prior accumulations of amino acids. We re-emphasize the uphill operation of System L, and by prior depletion of cellular amino acids show that this system must receive energy beyond that made available by their coupled exodus. After this depletion the Na+-indepdendent accumulation of the norbornane amino acid, 2-aminobicycloheptane-2-carboxylic acid becomes strongly subject to stimulation by incubation with glucose. Energy transfer between Systems A and L through the mutual substrate action of ordinary amino acids was minimized although not entirely avoided by the use of amino acid analogs specific to each system. When 2,4-dinitrophenol was included in the depleting treatment, and pyruvate, phenazine methosulfate, or glucose used for restoration, recovery of uptake of the norbornane amino acid was independent of external Na+ or K+ levels. Restoration or the uptake of 2-(methylamino)isobutyric acid was, however, decreased by omission of external K+. Contrary to an earlier finding, restoration of uptake of each of these amino acids was associated with distinct and usually correlated rises in cellular ATP levels. ATP addition failed to stimulate exodus of the norbornane amino acid from plasma membrane vesicles, although either NADH or phenazine methosulfate did stimulate exodus. ATP production and use is thus associated with transport energization although evidence for a direct role failed to appear.

Association of hepatic system A amino acid transporter with the membrane-cytoskeletal proteins ankyrin and fodrin.

System A activity is a highly regulated mechanism for the active transport of zwitterionic amino acids into mammalian cells. Monoclonal antibodies generated against a previously unidentified rat liver plasma membrane-associated protein were shown to immunoprecipitate solubilized System A transport activity. The immunoreactive protein was later determined by immunoblotting and peptide microsequencing to be rat liver alpha-fodrin (non-erythroid spectrin). Antibody against ankyrin, a protein that often serves as a bridge between integral membrane proteins and fodrin, also immunoprecipitated System A transport activity. Fractionation of solubilized plasma membrane proteins on sucrose gradients revealed that the System A transporter co-migrated as a complex with fodrin and ankyrin, even in the presence of detergent and urea. In contrast, the System N amino acid transporter does not co-migrate with ankyrin and fodrin, nor does the anti-fodrin antibody immunoprecipitate System N activity. The present data are the first to demonstrate an association between an organic solute transporter and the membranocytoskeletal proteins ankyrin and fodrin.

Substrate-dependent adaptive regulation and trans-inhibition of System A-mediated amino acid transport. Studies using rat hepatoma plasma membrane vesicles.

Substrate-dependent regulation of amino acid transport by System A occurs by both direct action at the carrier (trans-inhibition) and transcriptional control (adaptive regulation). While experiments with intact cells have led to working models that describe these regulatory phenomena, the use of subcellular approaches will serve to refine the present hypotheses. Adaptive induction of System A transport following amino acid starvation of cells was shown to be dependent on de novo RNA and protein synthesis, and the stimulated activity was shown to be retained in isolated plasma membrane vesicles. This stimulated transport activity was tightly associated with the plasma membrane, but could be solubilized by 4 M urea and 2.5% cholate, and recovered following reconstitution of the protein into artificial proteoliposomes. These data support the working hypothesis that adaptive induction of transport is the result of de novo synthesis and insertion into the plasma membrane of System A carrier protein. In contrast, the activity of System ASC in the vesicles from the amino acid starved cells was actually reduced by 2-5-fold when compared to amino acid-fed cells. A more rapid form of regulation of System A activity is trans-inhibition. The use of isolated plasma membrane vesicles demonstrated that trans-inhibition in whole cells did not survive membrane isolation. However, substrate loading of isolated membrane vesicles containing high levels of System A activity, produced trans-inhibition in a very specific manner in that System A substrates resulted in decreased transport activity, while those amino acids which are poor substrates for the System A carrier did not. Thus, trans-inhibition is not the result of a recycling process involving an intracellular pool of carriers, but rather can be accounted for by differences in the kinetics for amino acid binding and/or translocation on the two sides of the membrane.

Expressed human hippocampal ASCT1 amino acid transporter exhibits a pH-dependent change in substrate specificity.

In mammalian cells, the basal Na+-dependent uptake for many of the neutral amino acids is mediated by a transport activity designated System ASC. A cloned human brain cDNA sequence, ASCT1, encodes a Na+-dependent neutral amino acid transport activity that exhibits a substrate specificity similar to that commonly associated with System ASC. However, the characteristics of ASC activity varies significantly between cell types and not all tissues contain detectable levels of ASCT1 mRNA. A unique property of System ASC activity is an altered substrate selectivity such that at pH values below 7.4 anionic amino acids function as inhibitors and substrates. The experiments in this report were designed to determine if the cloned ASCT1 transporter exhibited this pH-dependent anionic transport. Following transfection of HeLa cells with the ASCT1 cDNA, transport strongly favored neutral zwitterionic) amino acids when uptake was measured at a physiologic pH value of 7.5. However, lowering the assay pH to 5.5 significantly enhanced the interaction of the ASCT1 carrier with anionic amino acids such as cysteate, in a pH-dependent manner. The apparent pK for the titratable group was in the range of 6.5-7.0. These results provide evidence that the human brain ASCT1 transporter exhibits the most distinguishing characteristic known for System ASC and provides a model system to investigate the molecular basis for this shift in substrate acceptance.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

Placental anionic and cationic amino acid transporter expression in growth hormone overexpressing and null IGF-II or null IGF-I receptor mice.

The role of growth hormone (GH), insulin-like growth factor (IGF)-II and the IGF-I receptor (IGF-Ir) in the regulation of the in vivo expression of Na(+)-coupled anionic [System X-AG; GLAST1 (EAAT1), GLT1 (EAAT2), EAAC1 (EAAT3), EAAT4; where the human homologues of amino acid transport proteins first cloned in the rat are given in parentheses] and Na(+)-independent cationic (System y(+);CAT1) amino acid transport proteins was evaluated by comparing transporter expression in day 17 placentae of mice that overexpressed bovine GH (GH+) or that carried null gene mutations for IGF-II or IGF-Ir. Northern analysis revealed no apparent difference in the mRNA content of GLAST1 (EAAT1), EAAC1 (EAAT3), or EAAT4, in homogenates of GH+ placentae, but levels of GLT1 (EAAT2) and CAT1 mRNA were increased. Immunoblot analysis revealed that whole-placental steady-state GLAST1 (EAAT1), EAAC1 (EAAT3), and EAAT4 protein levels were not affected by GH+, whereas GLT1 (EAAT2) levels were increased. Immunohistochemical analysis showed that the cell-specific expression of the anionic and CAT1 transporters was not affected by overexpression of GH. Similar analyses of null IGF-II placentae demonstrated increases in GLAST1 (EAAT1), EAAT4 and CAT1 mRNAs. Parallel immunoblot analysis demonstrated decreased expression of GLT1 (EAAT2), GLAST1 (EAAT1) and EAAC1 (EAAT3) protein, but an increased expression of EAAT4. In null IGF-II and IGF-Ir placentae, however, GLT1 (EAAT2) and EAAC1 (EAAT3) protein content was decreased in junctional zone cells, whereas CAT1 content was increased in junctional and labyrinth zone cells. These data indicate that an excess level of GH stimulates GLT1 (EAAT2) expression and that a normal level of IGF-II is required for typical expression of GLT1 (EAAT2), GLAST1 (EAAT1) and EAAC1 (EAAT3), but that IGF-II downregulates the expression of EAAT4 and CAT1.

Demonstration of system y+L activity on the basal plasma membrane surface of rat placenta and developmentally regulated expression of 4F2HC mRNA.

Na(+)-independent cationic amino acid transport in the rat placenta occurs by leucine-sensitive and leucine-insensitive pathways. The ontogeny of these transport mechanisms within the rat placenta has been described recently. To assign the leucine-inhibitable portion of uptake definitively the uptake of [3H]arginine was studied in the presence of both BCH (to inhibit system Bo,+) and varied concentrations of leucine. Uptake of arginine into basal-enriched membrane vesicles derived from rat placenta was, in the presence of sodium, inhibited by micromolar concentrations of leucine, consistent with assignment of this activity to system y+L. In contrast, the majority of arginine uptake into apical-enriched membrane vesicles was leucine insensitive. Messenger RNA derived from rat placenta at days 14, 16, 18 and 20 of gestation was hybridized with full-length rat cDNA probes against NBAT and 4F2HC (thought to encode proteins associated with system bo,+ and y+L activities, respectively). No NBAT mRNA was detected, whereas 4F2HC mRNA was present at all gestational stages, increasing 12-fold over the last third of gestation. It is concluded that system y+L is present in the basal plasma membrane of the rat placenta syncytium and is subject to developmental regulation by a mechanism that alters the steady content of 4F2HC mRNA.

Amino acid-dependent inactivation of glucagon-induced System A transport activity in cultured rat hepatocytes.

Hepatocytes isolated from glucagon-treated rats contain stimulated System A activity. If these cells are placed in primary culture, the enhanced transport decays rapidly provided the culture medium contains substrate amino acids. This amino acid-dependent inactivation can be composed of trans-inhibition (protein synthesis-independent), repression (protein synthesis-dependent), or both depending on the particular substrate tested. Repression was most prominently observed with a group of small neutral amino acids that are commonly found in proteins. A strong trans-inhibition response was induced by a variety of amino acid analogs. Amino acids showing no reactivity with System A produced neither trans-inhibition nor repression. Repression of System A activity in culture was blocked by inhibitors of both RNA and protein synthesis. In contrast to inhibitors of RNA biosynthesis such as actinomycin and alpha-amanitin, inhibitors of poly(A) polymerase (cordycepin and adenine-9-beta-D-arabinopyranoside) did not prevent the inactivation of the transport activity. These results demonstrate that both the stimulation of activity and the turnover of the hepatic System A activity are controlled at the transcriptional level.

Ontogeny and localization of the neutral amino acid transporter ASCT1 in rat brain.

ASCT1 is a protein that encodes System ASC, a sodium-dependent amino acid transport activity that transports primarily zwitterionic amino acids at physiological pH. ASCT1 has a 39-44% identity to the EAAT family of glutamate transporters. At extracellular pH values below 7.4, ASCT1 shifts substrate specificity to transport anionic amino acids. In this study we have examined the location of the ASCT1 transporter by immunohistochemistry in the developing rat brain. In addition, we have examined the cellular localization of ASCT1 in glial and neuronal cultures. The presence of ASCT1 immunoreactivity (ASCT1ir) in the developing brain was detectable as early as 14 days of gestation. At the cellular level, ASCT1ir was prominent in hippocampal pyramidal and dentate granule neurons. In the cerebellum, Purkinje cells and their dendrites were intensely labeled, whereas the granule and molecular layers were moderately labeled. In the cerebral cortex, neuronal cell bodies in all lamina and scattered astrocytes showed intense ASCT1ir. Double labeling experiments in vitro confirmed that ASCT1 was localized to both glia and neurons. These data illustrate that the rat ASCT1 transporter is expressed in the developing brain at levels equivalent to those observed in adult tissue. In addition, the expression and localization of ASCT1 are consistent with its possible role in pathophysiological processes that involve glutamate toxicity.

An example of nutrient control of gene expression: amino acid-dependent regulation of asparagine synthetase.

Amino acid deprivation of mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities, among these is included asparagine synthetase (AS). A full length cDNA clone for rat AS was isolated previously from a subtracted cDNA library enriched for amino acid-regulated sequences. The present report summarizes the use of the AS cDNA to investigate the amino acid-dependent regulation of AS mRNA in normal rat liver and Fao hepatoma cells. In response to complete amino acid starvation, there was an increase in steady state AS mRNA content. Three species of mRNA, approximately 2.0, 2.5 and 4.0 kb, were detected and each was simultaneously regulated to the same degree. In hepatoma cells the increased AS mRNA content was prevented by either actinomycin D or cycloheximide. Partial repression of the AS mRNA content was maintained by the presence of a single amino acid in the culture medium, but the effectiveness varied. Glutamine effectively repressed the AS mRNA content, even at a concentration 10 times below its plasma level. Conversely, depletion of selected single amino acids from complete culture medium also caused up-regulation. A role for tRNA charging in the signalling mechanism was suggested by the observation that the addition of histidinol, an inhibitor of histidinyl tRNA synthetase, caused an increase in AS mRNA content when added to complete medium. The increased AS mRNA is associated with polysomes and is actively translated. The data indicate that nutrient regulation of the rat AS gene occurs by a general control mechanism that is responsive to the availability of selected individual amino acids.

Characteristics and regulation of hepatic glutamine transport.

Glutamine is an important amino acid because of its key role in the transfer of both carbon and nitrogen between tissues in the body. Specific tissues are usually associated with either net synthesis or net utilization of glutamine, but the liver plays a central role in glutamine homeostasis, in that it can shift to function in either capacity. This capability, along with the localization of urea biosynthesis in the periportal hepatocytes, focuses attention on the transport mechanisms in hepatocytes for uptake and release of glutamine. Active transport of glutamine by hepatocytes is mediated by a Na(+)-dependent activity termed system N, which exhibits a rather narrow substrate specificity mediating uptake of histidine and asparagine as well as of glutamine. This secondary active transport system allows for the net accumulation of glutamine against a concentration gradient and maintenance of intracellular concentrations of glutamine between 4 and 8 mM in the face of a plasma concentration of 0.6 mM. Utilization of the Na+ electrochemical gradient as a driving force ensures that the system N carrier catalyzes a unidirectional transport event favoring the cytoplasm. It is obvious from the glutamine gradient across the plasma membrane that efflux of this amino acid is typically slower than accumulation; measurement of saturable, Na(+)-independent glutamine transport by system L substantiates this proposal. However, it is clear that under certain metabolic conditions the liver represents a source of glutamine for other tissues in the body and net efflux must occur. The system N transport activity in hepatocytes is regulated by hormones such as insulin, glucagon, and glucocorticoids, as demonstrated both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.