Multiplicity of the Amino Acid Permeases in Saccharomyces cerevisiae IV. Evidence for a General Amino Acid Permease

Kinetic and genetic evidences are presented to show that, in addition to specific amino acid permeases, Saccharomyces cerevisiae has a general amino acid permease which catalyzes the transport of basic and neutral amino acids, but most probably not that of proline. The general amino acid permease appears to be constitutive, and its activity is inhibited when ammonium ions are added to the culture medium. A mutant which has lost the general amino acid permease activity was isolated. Its mutation, named gap (general amino acid permease), is not allelic to the aap (amino acid permease) mutation of Surdin et al., which has a quite different phenotype and cannot be considered as having selectively lost the general amino acid permease activity.     

Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family

Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra(Rl)). Characterization of the solute specificity of Bra(Rl) shows it to be the second general amino acid permease of R. leguminosarum. Although Bra(Rl) has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be alpha-amino acids. Consistent with this, Bra(Rl) is the first ABC transporter to be shown to transport gamma-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by Bra(Rl) does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine. Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraC(Rl). Therefore, a second, unidentified solute binding protein may interact with the BraDEFG(Rl) membrane complex during L-alanine uptake. Overall, the data indicate that Bra(Rl) is a general amino acid permease of the HAAT family. Furthermore, Bra(Rl) has the broadest solute specificity of any characterized bacterial amino acid transporter.     

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.     

Differential expression of two related amino acid transporters with differing substrate specificity in Arabidopsis thaliana

A general amino acid permease cDNA ( AAP2 ) was isolated from Arabidopsis by complementation of a yeast mutant defective in citrulline uptake. Direct transport measurements in yeast show that the protein mediates uptake of l -[¹⁴C]-citrulline and l -[¹⁴C]-proline. Detailed analyses of the substrate specificity by competition studies demonstrate that all proteogenic amino acids are recognized by the carrier, including those that represent the major transport forms of reduced nitrogen in many species, i.e. glutamine, glutamate and asparagine. Thus, AAP2 is less selective as compared with AAP1 and transports basic amino acids such as histidine as shown by expression in a histidine transport-deficient yeast strain. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows significant homologies to the Arabidopsis broad specificity permease AAP1, and a limited homology to bacterial branched chain amino acid transporters, but not to any other known proteins. Alterations in the charged residues as compared with AAP1 in four regions might be involved in the difference in selectivity towards basic amino acids. Both genes are highly expressed in developing pods indicating a role in supplying the developing seeds with reduced nitrogen. AAP2 is selectively expressed in the stem and might therefore play a role in xylem-to-phloem transfer of amino acids during seed filling. Furthermore in situ hybridization shows that both genes are expressed in the vascular system of cotyledons in developing seedlings.     

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.     

Enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media

The enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-L-Lys(Ac)-D-Ala-D-Lac-OH and Ac-D-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the D-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%.     

Amino acid transport in taxonomically diverse cyanobacteria and identification of two genes encoding elements of a neutral amino acid permease putatively involved in recapture of leaked hydrophobic amino acids.

The activities of uptake of thirteen 14C-labeled amino acids were determined in nine cyanobacteria, including the unicellular strains Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803; the filamentous strain Pseudanabaena sp. strain PCC 6903, and the filamentous, heterocyst-forming strains Anabaena sp. strains PCC 7120 and PCC 7937; Nostoc sp. strains PCC 7413 and PCC 7107; Calothrix sp. strain PCC 7601 (which is a mutant unable to develop heterocysts); and Fischerella muscicola UTEX 1829. Amino acid transport mutants, selected as mutants resistant to some amino acid analogs, were isolated from the Anabaena, Nostoc, Calothrix, and Pseudanabaena strains. All of the tested cyanobacteria bear at least a neutral amino acid transport system, and some strains also bear transport systems specific for basic or acidic amino acids. Two genes, natA and natB, encoding elements (conserved component, NatA, and periplasmic binding protein, NatB) of an ABC-type permease for neutral amino acids were identified by insertional mutagenesis of strain PCC 6803 open reading frames from the recently published genomic DNA sequence of this cyanobacterium. DNA sequences homologous to natA and natB from strain PCC 6803 were detected by hybridization in eight cyanobacterial strains tested. Mutants unable to transport neutral amino acids, including natA and natB insertional mutants, accumulated in the extracellular medium a set of amino acids that always included Ala, Val, Phe, Ile, and Leu. A general role for a cyanobacterial neutral amino acid permease in recapture of hydrophobic amino acids leaked from the cells is suggested.     

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Physiological and regulatory properties of the general amino acid transport system of Neurospora crassa.

The fundamental properties of the general amino acid transport system of Neurospora crassa were investigated in the conidial stage of the life cycle. The transport activity was found to be under genetic control, and an isogenic set of mutants deficient for the neutral, basic, or general amino acid transport systems and combinations thereof was constructed and used for analyzing the properties specific to the general permease. Amino acid transport by this system was found to be a carrier-mediated active process with broad specificity for the neutral and basic amino acids. Kinetic analysis revealed that a common binding site functioned to transport both neutral and basic amino acids and that the permease had a high affinity for its substrates. The kinetic parameters Km, Vmax, and Ki were defined for several substrates. Two modes of regulation were detected: substrate inhibition and ammonium repression. Activity of the general system was enhanced by the removal of ammonium ions from the incubation medium with a concomitant decline in either neutral or basic permease activity, suggesting that a common component exists between the neutral and the general systems and between the basic and the general systems.     

Purification and characterisation of glucose (xylose) isomerase from Chainia sp. (NCL 82-5-1)

Glucose (xylose) isomerase is an important enzyme in high fructose syrup industry. The enzyme generally occurs intracellularly and is specific for both glucose and xylose. A rare actinomycete Chainia sp. (NCL 82-5-1) produces extracellular specific glucose and xylose isomerases and an intracellular glucose (xylose) isomerase. The intracellular enzyme is isolated by cell autolysis and purified by preparative polyacrylamide gel electrophoresis. Its properties are studied and compared with those of extracellular specific xylose isomerase. The intracellular enzyme has a molecular weight of 1,58,000 daltons with four equal subunits of 40,700 daltons. The N-terminal amino acid sequence analysis shows Arg at the N-terminal. Diethylpyrocarbonate inhibited the enzyme and the inhibition kinetics study shows the presence of at least 2 essential His residues. The amino acid analysis shows the absence of Cys and a high proportion of hydrophobic and acidic amino acids.     

Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum.

The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (delta psi) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H+ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H+-arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.     

Influence of hydrogenothrophic methane formation on the thermophilic anaerobic degradation of protein and amino acids

Abstract Thermophilic (55°C) protein (peptone) degradation was studied in steady state, laboratory-scale reactors. Peptone was easily hydrolysed to amino acids under methanogenic conditions, and all amino acids were completely degraded to volatile fatty acids, carbon dioxide and ammonium. Under these conditions, amino acids known to be oxidatively deaminated were degraded more slowly than the other amino acids. Inhibition of methanogenesis by 2-bromoethanesulfonic acid led to the accumulation of hydrogen in the gas phase and to the immediate inhibition of both protein hydrolysis and the degradation of amino acids that are preferentially oxidatively deaminated. These effects resulted in lower concentrations of all volatile fatty acids except for butyrate and caproate, which increased in concentration. Interspecies hydrogen transfer appeared to be necessary for the complete degradation of alanine, phenylalanine, methionine, valine, leucine and isoleucine. α-Aminobutyrate also accumulated when methanogenesis was inhibited.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

Ureidosuccinic acid permeation in Saccharomyces cerevisiae.

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20-25 mumol/ml cell water per min. The apparent Km is 3-10(-5) M. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyldrazone (CCCP) or NaN3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an alpha-N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.     

Comparative physiologial studies of the yeast and mycelial forms of Histoplasma capsulaum: uptake and incorporation of L-leucine

l-Leucine entered the cells of both morphological forms of Histoplasma capsulatum by a permease-like system at low external concentrations of substrate. However, at levels greater than 5 x 10(-5)m l-leucine, the amino acid entered the cells both through a simple diffusion-like process and the permease-like system. The rate of the amino acid diffusion into yeast and mycelial forms appeared to be the same, whereas the initial rate of accumulation through the permease-like system was 5 to 10 times faster in the mycelial phase than it was in the yeast phase. The Michaelis constants were 2.2 x 10(-5)m in yeast phase and 2 x 10(-5)m in mycelial phase cells. Transport of l-leucine at an external concentration of 10(-5)m showed all of the characteristics of a system of active transport, which was dependent on temperature and pH. Displacement or removal of the alpha-amino group, or modification of the alpha-carboxyl group abolished amino acid uptake. The process was competitively inhibited by neutral aliphatic side-chain amino acids (inhibition constants ranged from 1.5 x 10(-5) to 6.2 x 10(-5)m). Neutral aromatic side-chain amino acids and the d-isomers of leucine and valine did not inhibit l-leucine uptake. These data were interpreted to mean that the l-leucine transport system is stereospecific and is highly specific for neutral aliphatic side-chain amino acids. Incorporation of l-leucine into macromolecules occurred at almost the same rate in both morphological forms of the fungus. The mycelial phase but not the yeast phase showed a slight initial lag in incorporation. In both morphological forms the intracellular pool of l-leucine was of limited capacity, and the total uptake of the amino acid was a function of intracellular pool size. The initial rate of l-leucine uptake was independent of the level of intracellular pool. Both morphological forms deaminated and degraded only a minor fraction of the accumulated leucine.     

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.