Secondary Active Transport Research Articles

A Molecular Gearbox: The Mechanical And Regulatory Complexity Of The Vacuolar ATPase Revealed

Vacuolar H+-ATPases (V-ATPases) are ATP-driven rotary molecular motors that function as transmembrane proton pumps in all eukaryotic cells. Central roles of V-ATPases in cell physiology include: energising secondary active transport, maintaining acidity in intracellular compartments, and pumping acid out of the cell. Loss of V-ATPase function is associated with forms of kidney disease and inherited deafness; excess or unregulated activity is implicated in diseases such as osteoporosis and in tumour metastasis or multidrug resistance.

Diurnal expression of the rat intestinal sodium–glucose cotransporter 1 (SGLT1) is independent of local luminal factors

The intestinal sodium-glucose cotransporter 1 (SGLT1) is responsible for all secondary active transport of dietary glucose, and it presents a potential therapeutic target for obesity and diabetes. SGLT1 expression varies with a profound diurnal rhythm, matching expression to nutrient intake. The mechanisms entraining this rhythm remain unknown. We investigated the role of local nutrient signals in diurnal SGLT1 entrainment. Male Sprague-Dawley rats, which were acclimatized to a 12:12 light:dark cycle, underwent laparotomy with formation of isolated proximal jejunal loops (Thiry-Vella loops). Animals were recovered for 10 days before harvesting at 4 6-h intervals (Zeitgeber times ZT3, ZT9, ZT15, and ZT21, where ZT0 is lights on; n = 6-8). SGLT1 expression was assessed in protein, and mRNA extracts of mucosa were harvested from both isolated loops (LOOP) and remnant jejunum (JEJ). Isolated loops were healthy but atrophic with minimal changes to villus architecture. A normal anticipatory rhythm was observed in Sglt1 transcription in both LOOP and JEJ, with the peak signal at ZT9 (2.7-fold, P < .001). Normal diurnal rhythms were also observed in the protein signal, with peak expression in both LOOP and JEJ at ZT9 to 15 (2.1-fold, P < .05). However, an additional more mobile polypeptide band was also observed in all LOOP samples but not in JEJ samples (61 kDa vs 69 kDa). Enzymatic deglycosylation suggested this to be deglycosylated SGLT1. The persistence of SGLT1 rhythmicity in isolated loops indicates that diurnal induction is independent of local luminal nutrient delivery, and it suggests a reliance on systemic entrainment pathways. However, local luminal signals may regulate glycosylation and, therefore, the posttranslational handling of SGLT1.

MacB ABC Transporter Is a Dimer Whose ATPase Activity and Macrolide-binding Capacity Are Regulated by the Membrane Fusion Protein MacA

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

The sodium‐dependentd‐glucose transport protein ofHelicobacter pylori

Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.

Luminal Na(+)/H (+) exchange in the proximal tubule.

The proximal tubule is critical for whole-organism volume and acid-base homeostasis by reabsorbing filtered water, NaCl, bicarbonate, and citrate, as well as by excreting acid in the form of hydrogen and ammonium ions and producing new bicarbonate in the process. Filtered organic solutes such as amino acids, oligopeptides, and proteins are also retrieved by the proximal tubule. Luminal membrane Na(+)/H(+) exchangers either directly mediate or indirectly contribute to each of these processes. Na(+)/H(+) exchangers are a family of secondary active transporters with diverse tissue and subcellular distributions. Two isoforms, NHE3 and NHE8, are expressed at the luminal membrane of the proximal tubule. NHE3 is the prevalent isoform in adults, is the most extensively studied, and is tightly regulated by a large number of agonists and physiological conditions acting via partially defined molecular mechanisms. Comparatively little is known about NHE8, which is highly expressed at the lumen of the neonatal proximal tubule and is mostly intracellular in adults. This article discusses the physiology of proximal Na(+)/H(+) exchange, the multiple mechanisms of NHE3 regulation, and the reciprocal relationship between NHE3 and NHE8 at the lumen of the proximal tubule.

Pushing and Pulling the Cardiac Sodium/Hydrogen Exchanger

See related article, pages 881–890 The strangest animal in Dr Dolittle’s zoo was a Pushmi-pullyu (pronounced push-me-pull-you), a mythical chimera of 2 antelopes, with a head at each end. Sometimes it looked and moved forwards, sometimes backwards. The direction could be coaxed by Dr Dolittle (played originally by Rex Harrison in the Hollywood movie), who spoke to the animal in its own language. A report from the laboratory of Avkiran, published in this issue of Circulation Research ,1 shows that there is something strangely reminiscent of the Pushmi-pullyu in the cardiac sodium–hydrogen exchanger (NHE1), a ubiquitous, dimeric glycoprotein, intimately involved with the regulation of intracellular pH (pHi) (reviewed elsewhere2). In cardiac myocytes, the NHE1 protein product is expressed at the sarcolemma. It is a secondary active transporter expelling one H+ ion in exchange for the entry of one Na+ ion, and its activity is governed acutely by the intracellular H+ ion concentration (ie, pHi). Activity increases steeply when pHi falls from its resting level of ≈7.2. This is why the protein is so effective as a pHi regulator. But why the resemblance to a Pushmi-pullyu? It is becoming apparent that NHE1 in the cardiac cell can be both stimulated and inhibited directly by intracellular signaling cascades. Like Dr Dolittle, these talk to the transporter, and the chorus determines whether cellular H+ extrusion during intracellular acidosis is stimulated or inhibited. It has long been known that the pHi sensitivity of NHE1 can be enhanced through a coupling of the transport protein to surface membrane receptors, operating via endogenous intracellular ligands.2 Many of the pathways involved have been highlighted in other tissues, but, in the cardiac myocyte, typical extracellular receptor agonists that stimulate NHE1 activity are angiotensin II (acting …

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.

Ins and outs of major facilitator superfamily antiporters.

The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.

The ClC-0 chloride channel is a 'broken' Cl−/H+ antiporter

Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC ‘chloride channel’ family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a ‘broken’ Cl−/H+ antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.

Reduced V-ATPase Activity in thetrans-Golgi Network Causes Oxylipin-Dependent Hypocotyl Growth Inhibition inArabidopsis

Regulated cell expansion allows plants to adapt their morphogenesis to prevailing environmental conditions. Cell expansion is driven by turgor pressure created by osmotic water uptake and is restricted by the extensibility of the cell wall, which in turn is regulated by the synthesis, incorporation, and cross-linking of new cell wall components. The vacuolar H(+)-ATPase (V-ATPase) could provide a way to coordinately regulate turgor pressure and cell wall synthesis, as it energizes the secondary active transport of solutes across the tonoplast and also has an important function in the trans-Golgi network (TGN), which affects synthesis and trafficking of cell wall components. We have previously shown that det3, a mutant with reduced V-ATPase activity, has a severe defect in cell expansion. However, it was not clear if this is caused by a defect in turgor pressure or in cell wall synthesis. Here, we show that inhibition of the tonoplast-localized V-ATPase subunit isoform VHA-a3 does not impair cell expansion. By contrast, inhibition of the TGN-localized isoform VHA-a1 is sufficient to restrict cell expansion. Furthermore, we provide evidence that the reduced hypocotyl cell expansion in det3 is conditional and due to active, hormone-mediated growth inhibition caused by a cell wall defect.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).

A High-Capacity Membrane Potential FRET-Based Assay for the Sodium-Coupled Glucose Co-transporter SGLT1

Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.

Novel Ouabain Receptors in Bovine Adrenal Cortex: Evidence for Ouabain Transport

Novel high affinity binding sites for ouabain (NOR) are present in bovine adrenal cortex (Ward SC, Hypertension 2002; 39[pt. 2]:536). To distinguish NOR from classical sodium‐potassium pumps (Na pumps), we used fresh adrenal cortical membranes. Scatchard analysis showed that K+ (20mM) reduced the apparent Kd for ouabain binding to Na pumps from 4.6 ± 0.2 to 240 ± 7.2 nM with no change in Bmax (10.1 ± 0.1 vs. 10.3 ± 0.2 pmoles/mg). K+ increased the Kd for ouabain binding to NOR and increased the Bmax (0.15 ± 0.02 vs. 0.26 ± 0.06 pmoles/mg). Moreover, the effect of Na+ was more dramatic; Na+ inhibited ouabain binding to Na pumps with no change in Bmax, while Na+ increased the apparent Kd for ouabain binding to NOR and increased Bmax &gt; 25‐fold. Ouabain binding to the Na pump in stored membranes remained constant for up to 50 days whereas NOR related binding declined with a t1/2 of 6.8 days. The association of ouabain with membranes was 0.538 pmoles/mg when [Na+]o » [Na+]i, and was 0.152 pmoles/mg when [Na+]o « [Na+]i, and was sensitive to permeable anions. NOR binding was sensitive to osmolarity. Photoactive analogs of ouabain labeled a 35 kDa polypeptide. We conclude that NOR mediates the secondary active sodium‐dependent transport of 3H‐ouabain into sealed membrane vesicles and that this process is electrogenic. NOR may mediate the secretion of cardiac glycosides by the adrenal cortex in vivo. Supported by HL078870 and HL075584.

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-007-9287-z) contains supplementary material, which is available to authorized users.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.

The nucleobase–ascorbate transporter (NAT) family: genomics, evolution, structure–function relationships and physiological role

This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.

Neuronal Glutamate Transporters Vary in Substrate Transport Rate but Not in Unitary Anion Channel Conductance

Excitatory amino acid transporters (EAATs) not only sustain a secondary active glutamate transport but also function as anion-selective ion channels. The relative proportion of currents generated by glutamate transport or by the chloride conductance varies for each cloned EAAT subtype. For EAAT1, EAAT2, and EAAT3, the anion current is only a small component of the total transporter-associated current amplitude, whereas EAAT4 and EAAT5 transporters mediate predominantly anion currents. We here demonstrate that the distinct current proportions are entirely due to differences in glutamate transport rates. EAAT3 and EAAT4 differ in unitary glutamate transport rates as well as in the voltage and substrate dependence of anion channel opening, but ion conduction properties are very similar. Noise analysis revealed identical unitary current amplitudes and similar absolute open probabilities for the two anion channels. The low glutamate transport rate of EAAT4 allows regulation of cellular excitability without interfering with extracellular glutamate homeostasis and makes this EAAT isoform ideally suited to regulate excitability in dendritic spines of Purkinje neurons.

Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT.

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparent substrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward- and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.

Novel subsets of the Arabidopsis plasmalemma phosphoproteome identify phosphorylation sites in secondary active transporters

The generation of novel subsets of phosphorylation sites is needed to complement the present Arabidopsis plasma membrane phosphoprotein repertoire, where several families of proteins are under-represented. In this work, different combinations of chromatographic steps were first compared for capacity to resolve model phosphopeptides before characterisation from PSD fragments in MALDI MS/MS. Nearly half of the phosphorylation sites detected in the Arabidopsis plasmalemma using the optimised procedure were novel, and two-thirds of protein accessions identified secondary active transporters. These included phosphate/H + symporters, ammonium and nitrate transporters, different alkali cation exchangers, a urea/H + symporter, a glucose transporter, a purine permease, and peptide transporters. There has been previous functional evidence for phosphorylation of only a minority of these, the regulation of others having been essentially investigated at the transcriptional level. The demonstration of active phosphorylation sites in such a diverse set of secondary transporter families suggests that this regulation level plays a major role in the response of plants to nutrient availability.