Amino Acid Permease Research Articles

Pmr1, a Golgi Ca2+/Mn2+-ATPase, is a regulator of the target of rapamycin (TOR) signaling pathway in yeast.

The rapamycin.FKBP12 complex inhibits target of rapamycin (TOR) kinase in TORC1. We screened the yeast nonessential gene deletion collection to identify mutants that conferred rapamycin resistance, and we identified PMR1, encoding the Golgi Ca2+/Mn2+ -ATPase. Deleting PMR1 in two genetic backgrounds confers rapamycin resistance. Epistasis analyses show that Pmr1 functions upstream from Npr1 and Gln-3 in opposition to Lst8, a regulator of TOR. Npr1 kinase is largely cytoplasmic, and a portion localizes to the Golgi where amino acid permeases are modified and sorted. Nuclear translocation of Gln-3 and Gln-3 reporter activity in pmr1 cells are impaired, but expression of functional Gap1 in the plasma membrane of a pmr1 strain in response to nitrogen limitation is enhanced. These two phenotypes suggest up-regulation of Npr1 function in the absence of Pmr1. Together, our results establish that Pmr1-dependent Ca2+ and/or Mn2+ ion homeostasis is necessary for TOR signaling.

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.

Cloning and Sequencing of the Histidine Decarboxylase Gene from Photobacterium phosphoreum and its Functional Expression in Escherichia coli

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10°C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5′-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.

Activity-dependent Reversible Inactivation of the General Amino Acid Permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.

Successful Transporter Gets an EGO Boost

The trafficking of nutrient transporters is highly regulated and controlled by a variety of signal transduction pathways. A recent study by in Nature Cell Biology demonstrates a role for a novel GTPase-containing complex that allows the amino acid permease Gap1 to recycle out of late endosomal compartments. This complex is likely to play critical roles in allowing animal cells to mobilize their nutrient transporters in response to metabolic demands.

Arabidopsis LHT1 Is a High-Affinity Transporter for Cellular Amino Acid Uptake in Both Root Epidermis and Leaf Mesophyll

Amino acid transport in plants is mediated by at least two large families of plasma membrane transporters. Arabidopsis thaliana, a nonmycorrhizal species, is able to grow on media containing amino acids as the sole nitrogen source. Arabidopsis amino acid permease (AAP) subfamily genes are preferentially expressed in the vascular tissue, suggesting roles in long-distance transport between organs. We show that the broad-specificity, high-affinity amino acid transporter LYSINE HISTIDINE TRANSPORTER1 (LHT1), an AAP homolog, is expressed in both the rhizodermis and mesophyll of Arabidopsis. Seedlings deficient in LHT1 cannot use Glu or Asp as sole nitrogen sources because of the severe inhibition of amino acid uptake from the medium, and uptake of amino acids into mesophyll protoplasts is inhibited. Interestingly, lht1 mutants, which show growth defects on fertilized soil, can be rescued when LHT1 is reexpressed in green tissue. These findings are consistent with two major LHT1 functions: uptake in roots and supply of leaf mesophyll with xylem-derived amino acids. The capacity for amino acid uptake, and thus nitrogen use efficiency under limited inorganic N supply, is increased severalfold by LHT1 overexpression. These results suggest that LHT1 overexpression may improve the N efficiency of plant growth under limiting nitrogen, and the mutant analyses may enhance our understanding of N cycling in plants.

Global Analysis of Protein Palmitoylation in Yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Asi1 is an inner nuclear membrane protein that restricts promoter access of two latent transcription factors

Stp1 and Stp2 are homologous transcription factors in yeast that are synthesized as latent cytoplasmic precursors with NH2-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane–localized Ssy1–Ptr3–Ssy5 (SPS) sensor endoproteolytically processes Stp1 and Stp2, an event that releases the regulatory domains. The processed forms of Stp1 and Stp2 efficiently target to the nucleus and bind promoters of amino acid permease genes. In this study, we report that Asi1 is an integral component of the inner nuclear membrane that maintains the latent characteristics of unprocessed Stp1 and Stp2. In cells lacking Asi1, full-length forms of Stp1 and Stp2 constitutively induce SPS sensor–regulated genes. The regulatory domains of Stp1 and Stp2 contain a conserved motif that confers Asi1-mediated control when fused to an unrelated DNA-binding protein. Our results indicate that latent precursor forms of Stp1 and Stp2 inefficiently enter the nucleus; however, once there, Asi1 restricts them from binding SPS sensor–regulated promoters. These findings reveal an unanticipated role of inner nuclear membrane proteins in controlling gene expression.

A conserved GTPase-containing complex is required for intracellular sorting of the general amino-acid permease in yeast

The Saccharomyces cerevisiae general amino-acid permease, Gap1p, is a model for membrane proteins that are regulated by intracellular sorting according to physiological cues set by the availability of amino acids. Here, we report the identification of a conserved sorting complex for Gap1p, named the GTPase-containing complex for Gap1p sorting in the endosomes (GSE complex), which is required for proper sorting of Gap1p from the late endosome for eventual delivery to the plasma membrane. The complex contains two small GTPases (Gtr1p and Gtr2p) and three other proteins (Ybr077c, Ykr007w and Ltv1p) that are located in the late endosomal membrane. Importantly, Gtr2p interacts with the carboxy (C)-terminal cytosolic domain of Gap1p and a tyrosine-containing motif in this domain is necessary both to bind Gtr2p and to direct sorting of Gap1p to the plasma membrane. Together, these studies provide evidence that the GSE complex has a key role in trafficking Gap1p out of the endosome and may serve as coat proteins in this process.

Identification and Functional Characterization of theLactococcus lactisCodY-Regulated Branched-Chain Amino Acid Permease BcaP (CtrA)

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.

Amino Acids Regulate Retrieval of the Yeast General Amino Acid Permease from the Vacuolar Targeting Pathway

Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

The product of the SHR3 orthologue of Aspergillus nidulans has restricted range of amino acid transporter targets

The shrA gene of Aspergillus nidulans codes for a structural and functional homologue of Shr3p, a yeast ER membrane protein, which plays a crucial role in the secretory pathway of yeast amino acid permeases. shrA is a single-copy gene, whose expression is early activated during germination of A. nidulans conidiospores. ShrA is localized in the ER of the fungal cells and partially complements the shr3Δ phenotype. Differently from Saccharomyces cerevisiae, where SHr3p is necessary for membrane localization of the majority of amino acid permeases, deletion of the shrA locus in A. nidulans impairs a limited number of amino acid uptake activities, including those responsible for proline and aspartate transport. Strongly reduced membrane levels of a PrnB-sGFP fusion in a shrAΔ background clearly suggest a direct role of ShrA in the topogenesis of the proline specific transporter.

Carbon Catabolite Repression Regulates Amino Acid Permeases in Saccharomyces cerevisiae via the TOR Signaling Pathway

We have identified carbon catabolite repression (CCR) as a regulator of amino acid permeases in Saccharomyces cerevisiae, elucidated the permeases regulated by CCR, and identified the mechanisms involved in amino acid permease regulation by CCR. Transport of l-arginine and l-leucine was increased by approximately 10-25-fold in yeast grown in carbon sources alternate to glucose, indicating regulation by CCR. In wild type yeast the uptake (pmol/10(6) cells/h), in glucose versus galactose medium, of l-[(14)C]arginine was (0.24 +/- 0.04 versus 6.11 +/- 0.42) and l-[(14)C]leucine was (0.30 +/- 0.02 versus 3.60 +/- 0.50). The increase in amino acid uptake was maintained when galactose was replaced with glycerol. Deletion of gap1Delta and agp1Delta from the wild type strain did not alter CCR induced increase in l-leucine uptake; however, deletion of further amino acid permeases reduced the increase in l-leucine uptake in the following manner: 36% (gnp1Delta), 62% (bap2Delta), 83% (Delta(bap2-tat1)). Direct immunofluorescence showed large increases in the expression of Gnp1 and Bap2 proteins when grown in galactose compared with glucose medium. By extending the functional genomic approach to include major nutritional transducers of CCR in yeast, we concluded that SNF/MIG, GCN, or PSK pathways were not involved in the regulation of amino acid permeases by CCR. Strikingly, the deletion of TOR1, which regulates cellular response to changes in nitrogen availability, from the wild type strain abolished the CCR-induced amino acid uptake. Our results provide novel insights into the regulation of yeast amino acid permeases and signaling mechanisms involved in this regulation.

A novel high‐affinity arginine transporter from the human parasitic protozoan Leishmania donovani

We describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani, the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans-membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a high-affinity arginine transporter (Km 1.9 microM). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine-related compounds. Using green fluorescence protein (GFP) fused to the N-terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP-LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host-parasite interaction.

The general amino acid permease of Rhizobium leguminosarum strain 3841 is negatively regulated by the Ntr system.

Cosmid-borne and chromosomal lacZ fusions to aapJ. aapQ and aapM were used to examine the nitrogen regulation of the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841. Transcription of the first gene of the operon (aapJ), which encodes the periplasmic binding protein, was 2-4-fold higher than aapQ and aapM, which encode the integral membrane proteins, under various growth conditions. This may be due to the presence of a putative stem loop in the intergenic region between aapJ and aapQ. All aap fusions were derepressed 3-5-fold after growth on glutamate as a nitrogen source, which effectively causes nitrogen limitation. An ntrC mutant was derepressed for transcription of the aap operon and had high rates of amino acid transport when grown on ammonia as the nitrogen source. Thus NtrC negatively regulates the aap operon, contrary to its usual role in positive gene activation. These results confirm that the aap-operon is subject to complex regulation involving both transcriptional and post-transcriptional factors.

GAP1 activity is dependent on cAMP in Saccharomyces cerevisiae.

General amino acid permease (GAP1) activity was evaluated in adenylate cyclase-deficient Saccharomyces cerevisiae to determine the effect of cAMP levels on GAP1 activity. Lowering cAMP concentrations in the culture media led to a decrease in the initial rates of L-citrulline uptake. Kinetics of the amino acid transport system showed a partial loss of transport capacity, with no apparent modifications in permease affinity.

Deletion of RTS1 , Encoding a Regulatory Subunit of Protein Phosphatase 2A, Results in Constitutive Amino Acid Signaling via Increased Stp1p Processing

In Saccharomyces cerevisiae, extracellular amino acids are sensed at the plasma membrane by the SPS sensor, consisting of the transporter homologue Ssy1p, Ptr3p, and the endoprotease Ssy5p. Amino acid sensing results in proteolytic truncation of the transcription factors Stp1p and Stp2p, followed by their relocation from the cytoplasm to the nucleus, where they activate transcription of amino acid permease genes. We screened a transposon mutant library for constitutively signaling mutants, with the aim of identifying down-regulating components of the SPS-mediated pathway. Three isolated mutants were carrying a transposon in the RTS1 gene, which encodes a regulatory subunit of protein phosphatase 2A. We investigated the basal activity of the AGP1 and BAP2 promoters in rts1delta cells and found increased transcription from these promoters, as well as increased Stp1p processing, even in the absence of amino acids. Based on our findings we propose that the phosphatase complex containing Rts1p keeps the SPS-mediated pathway down-regulated in the absence of extracellular amino acids by dephosphorylating a component of the pathway.

Brefeldin A decreases the activity of the general amino acid permease (GAP1) and the more specific systems for L-leucine uptake in Saccharomyces cerevisiae.

Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.

Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display

Citrus unshiu is freeze tolerant to − 10 °C when fully acclimated after exposure to cold, nonfreezing temperatures. To gain an understanding of its cold tolerance mechanism, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and quantitative relative RT-PCR were used to study gene expression under a gradual cold-acclimation temperature regime. Six up-regulated and two down regulated genes were identified based on their amino acid sequences. The identified proteins encoded by the up-regulated genes were: 14-3-3 protein, 40S ribosomal protein S23, putative 60S ribosomal protein L15, nucleoside diphosphate kinase III protein, regulator of chromosome condensation-like protein, and amino acid permease 6. The proteins encoded by the two down-regulated genes were: miraculin-like protein and beta-galactosidase. Their individual function has been briefly reviewed based on published information. In addition to the findings in this study, we compared the function of cold responsive genes of Poncirus trifoliata, a very cold hardy relative of Citrus species that is freeze tolerant to − 30 °C when fully acclimated, to the function of genes in the current study.

ABC‐type neutral amino acid permease N‐I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.