Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation

To counteract fluctuating nutrient environments, plants have evolved high- and low-affinity uptake systems. These two systems were traditionally thought to be genetically distinct, but, recently, two Arabidopsis transporters, AtKUP1 and CHL1, were shown to have dual affinities. However, little is known about how a dual-affinity transporter works and the advantages of having a dual-affinity transporter. This study demonstrates that, in the case of CHL1, switching between the two modes of action is regulated by phosphorylation at threonine residue 101; when phosphorylated, CHL1 functions as a high-affinity nitrate transporter, whereas, when dephosphorylated, it functions as a low-affinity nitrate transporter. This regulatory mechanism allows plants to change rapidly between high- and low-affinity nitrate uptake, which may be critical when competing for limited nitrogen. These results demonstrate yet another regulatory role of phosphorylation in plant physiology.     

Characterisation and expression of monosaccharide transporters in lupins, <Emphasis Type="Italic">Lupinus polyphyllus</Emphasis> and <Emphasis Type="Italic">L. albus</Emphasis>

Monosaccharide transporter (MST) genes of Lupinus polyphyllus and L. albus were cloned, expressed and characterised. The isolation and functional characterisation of a cDNA clone and its corresponding genomic clone of a sugar transporter from L. polyphyllus (LpSTP1) is reported. Phylogenetic comparison of the nucleic and amino acid sequences showed the highest similarity to the AtSTP1 gene from Arabidopsis thaliana, which encodes a high affinity sugar transporter. The similar topology as well as the substrate specificity and expression pattern of LpSTP1 encoded protein additionally support the high similarity to the AtSTP1 gene product. The 1,590 bp LpSTP1 cDNA clone was heterologously expressed in yeast resulting in a fully functional specific sugar transporter. This transformation restored the viability of a yeast deletion mutant, which is devoid of all intrinsic MSTs and thus unable to take up and grow on hexose-containing media. The LpSTP1 protein is postulated to be a high-affinity MST since it supported growth best on media containing 0.2% hexose. Tissue-specific expression of LaSTP1 in L. albus was assayed by real-time PCR, which revealed that the lupin STP1 is mainly expressed in flower buds, flowers and young leaves. The results suggest that the main role of LaSTP1 is to catalyse monosaccharide import in sink tissues to meet increased carbohydrate demand during plant development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

Phosphorylation of the yeast nitrate transporter Ynt1 is essential for delivery to the plasma membrane during nitrogen limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.     

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.     

A phosphorylation in the c-terminal auto-inhibitory domain of the plant plasma membrane H+-ATPase activates the enzyme with no requirement for regulatory 14-3-3 proteins

The plant plasma membrane H(+)-ATPase is regulated by an auto-inhibitory C-terminal domain that can be displaced by phosphorylation of the penultimate residue, a Thr, and the subsequent binding of 14-3-3 proteins. By mass spectrometric analysis of plasma membrane H(+)-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum plants and suspension cells, we identified a new phosphorylation site, Thr-889, in a region of the C-terminal domain upstream of the 14-3-3 protein binding site. This residue was mutated into aspartate or alanine, and the mutated H(+)-ATPases expressed in the yeast Saccharomyces cerevisiae. Unlike wild-type PMA2, which could replace the yeast H(+)-ATPases, the PMA2-Thr889Ala mutant did not allow yeast growth, whereas the PMA2-Thr889Asp mutant resulted in improved growth and increased H(+)-ATPase activity despite reduced phosphorylation of the PMA2 penultimate residue and reduced 14-3-3 protein binding. To determine whether the regulation taking place at Thr-889 was independent of phosphorylation of the penultimate residue and 14-3-3 protein binding, we examined the effect of combining the PMA2-Thr889Asp mutation with mutations of other residues that impair phosphorylation of the penultimate residue and/or binding of 14-3-3 proteins. The results showed that in yeast, PMA2 Thr-889 phosphorylation could activate H(+)-ATPase if PMA2 was also phosphorylated at its penultimate residue. However, binding of 14-3-3 proteins was not required, although 14-3-3 binding resulted in further activation. These results were confirmed in N. tabacum suspension cells. These data define a new H(+)-ATPase activation mechanism that can take place without 14-3-3 proteins.     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Npr1 Ser/Thr protein kinase links nitrogen source quality and carbon availability with the yeast nitrate transporter (Ynt1) levels

Ynt1, the single high affinity nitrate and nitrite transporter of the yeast Hansenula polymorpha, is regulated by the quality of nitrogen sources. Preferred nitrogen sources cause Ynt1 dephosphorylation, ubiquitinylation, endocytosis, and vacuolar degradation. In contrast, under nitrogen limitation Ynt1 is phosphorylated and sorted to the plasma membrane. We show here the involvement of the Ser/Thr kinase HpNpr1 in Ynt1 phosphorylation and regulation of Ynt1 levels in response to nitrogen source quality and the availability of carbon. In Δnpr1, Ynt1 phosphorylation does not take place, although Ynt1 ubiquitin conjugates increase. As a result, in this strain Ynt1 is sorted to the vacuole, from both plasma membrane and the later biosynthetic pathway in nitrogen-free conditions and nitrate. In contrast, overexpression of NPR1 blocks down-regulation of Ynt1, increasing Ynt1 phosphorylation at Ser-244 and -246 and reducing ubiquitinylation. Furthermore, Npr1 is phosphorylated in response to the preferred nitrogen sources, and indeed it is dephosphorylated in nitrogen-free medium. Under conditions where Npr1 is phosphorylated, Ynt1 is not and vice versa. We show for the first time that carbon starvation leads to Npr1 phosphorylation, whereas Ynt1 is dephosphorylated and degraded in the vacuole. Rapamycin prevents this, indicating a possible role of the target of rapamycin signaling pathway in this process. We concluded that Npr1 plays a key role in adapting Ynt1 levels to the nitrogen quality and availability of a source of carbon.     

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.     

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.     

Mutants at Ser277 of Xenopus cdc2 protein kinase induce oocyte maturation in the absence of the positive regulatory phosphorylation site Thr161.

The cdc2 protein kinase is an important regulatory protein for both meiosis and mitosis. Previously, we demonstrated that simultaneous mutation of Thr14-->Ala14 and Tyr15-->Phe15 in the Xenopus cdc2 protein results in an activated cdc2 mutant that induces maturation in resting oocytes. In addition, we confirmed the importance of the positive regulatory phosphorylation site, Thr161, by demonstrating that cdc2 mutants containing additional mutations of Thr161-->Ala161 or Glu161 are inactive in the induction of oocyte maturation. Here, we have analyzed the importance of an additional putative cdc2 phosphorylation site,Ser277. Single mutation of Ser277-->Asp277 or Ala277 had no effect on activity, and these mutants were unable to induce Xenopus oocyte maturation. However, the double mutant Ala161/Asp277 was capable of inducing oocyte maturation, suggesting that mutation of Ser277-->Asp277 could compensate for the mutation of Thr161-->Ala161. The Asp277 mutation could also compensate for the Ala161 mutation in the background of the activating mutations Ala14/Phe15. Although mutants containing the compensatory Ala161 and Asp277 mutations were capable of inducing oocyte maturation, these mutant cdc2 proteins lacked detectable in vitro kinase activity. Tryptic phosphopeptide mapping of mutant cdc2 protein and comparison with in vitro synthesized peptides indicated that Ser277 is not a major site of phosphorylation in Xenopus oocytes; however, we cannot rule out the possibility of phosphorylation at this site in a biologically active subpopulation of cdc2 molecules. The data presented here, together with prior reports of Ser277 phosphorylation in somatic cells, suggest an important role for Ser277 in the regulation of cdc2 activity. The regulatory role of Ser277 most likely involves its indirect effects on the nearby residue Arg275, which participates in a structurally important ion pair with Glu173, which lies in the same loop as Thr161 in the cdc2 protein.     

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase: mutagenesis at metal site 2

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3'(2')-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in V(max) by a factor of 1.1 x 10(4). Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.     

Identification and characterization of the ATP-binding site in human pancreatic glucokinase

The central role of human pancreatic glucokinase in insulin secretion and, consequently, in maintenance of blood glucose levels has prompted investigation into identification of ATP-binding site residues and examination of ATP- and glucose-binding interactions. Because glucokinase has been resistant to crystallization, computer generated homology models were developed based on the X-ray crystal structure of the COOH-terminal domain of human brain hexokinase 1 bound to glucose and ADP or glucose and glucose-6-phosphate. Human pancreatic glucokinase mutants were designed based upon these models and on ATPase domain sequence conservation to identify and characterize potential glucose and ATP-binding sites. Specifically, mutants Asp78Ala, Thr82Ala, Lys90Ala, Lys102Ala, Gly227Ala, Thr228Ala, Ser336Leu, Ser411Ala, and Ser411Leu were constructed, expressed, purified, and kinetically characterized under steady-state conditions. Compared to their respective wild type controls, several mutants demonstrated dramatic changes in V(max), cooperativity of glucose binding and S(0.5) for ATP and glucose. Results suggest a role for Asp78, Thr82, Gly227, Thr228, and Ser336 in ATP binding and indicate these residues are essential for glucose phosphorylation by human pancreatic glucokinase.     

A hexose transporter homologue controls glucose repression in the methylotrophic yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser(85)) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker's yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport.     

NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis

Heterologous complementation of yeast mutants has enabled the isolation of genes encoding several families of amino acid transporters. Among them, NTR1 codes for a membrane protein with weak histidine transport activity. However at the sequence level, NTR1 is related to rather non-specific oligopeptide transporters from a variety of species including Arabidopsis and to the Arabidopsis nitrate transporter CHL1. A yeast mutant deficient in oligopeptide transport was constructed allowing to show that NTR1 functions as a high affinity, low specificity peptide transporter. In siliques NTR1-expression is restricted to the embryo, implicating a role in the nourishment of the developing seed.