Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

Transport mechanism of the neuronal excitatory amino acid transporter

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report Cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing for the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which conserved glutamate mediates proton symport.

Mechanisms of DNA Uptake by Naturally Competent Bacteria

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

Secondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate “coupling”. We untangle the “proton-metal coupling” behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: ΔpH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.

Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p

The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.

Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.

A Member of the Sugar Transporter Family, Stl1p Is the Glycerol/H+Symporter inSaccharomyces cerevisiae

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

ABSTRACT Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport.

Metabolic costs induced by lactate in the toad Bufo marinus: new mechanism behind oxygen debt?

The mechanism of an increase in metabolic rate induced by lactate was investigated in the toad Bufo marinus. Oxygen consumption (V˙o 2) was analyzed in fully aerobic animals under hypoxic conditions (7% O2 in air), accompanied by measurements of catecholamines in the plasma, and was measured in isolated hepatocytes in vitro under normoxia by using specific inhibitors of lactate proton symport [α-cyano-4-hydroxycinnamate (CHC)] and sodium proton exchange (EIPA). The rise in metabolic rate in vivo can be elicited by infusions of hyperosmotic (previous findings) or isosmotic sodium lactate solutions (this study). Despite previous findings of reduced metabolic stimulation under the effect of adrenergic blockers, the increase inV˙o 2 in vivo was not associated with elevated plasma catecholamine levels, suggesting local release and effect. In addition to the possible in vivo effect via catecholamines, lactate induced a rise in V˙o 2 of isolated hepatocytes, depending on the concentration present in a weakly buffered Ringer solution at pH 7.0. No increase was found at higher pH values (7.4 or 7.8) or in HEPES-buffered Ringer solution. Inhibition of the Lac−-H+ transporter with α-CHC or of the Na+/H+ exchanger with EIPA prevented the increase in metabolic rate. We conclude that increasedV˙o 2 at an elevated systemic lactate level may involve catecholamine action, but it is also caused by an increased energy demand of cellular acid-base regulation via stimulation of Na+/H+ exchange and thereby Na+-K+-ATPase. The effect depends on entry of lactic acid into the cells via lactate proton symport, which is likely favored by low cellular surface pH. We suggest that these energetic costs should also be considered in other physiological phenomena, e.g., when lactate is present during excess, postexerciseV˙o 2.