Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Complementary and Overlapping Selectivity of the Two-Peptide Bacteriocins Plantaricin EF and JK

Plantaricin EF and JK are both two-peptide bacteriocins produced by Lactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Deltapsi) and pH gradient (DeltapH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.     

Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis

The bacterium Pseudomonas aeruginosa is commonly isolated from the general environment and also infects the lungs of patients with cystic fibrosis (CF). Iron in mammals is not freely available to infecting pathogens although significant amounts of extracellular iron are available in the sputum that occurs in the lungs of CF patients. P. aeruginosa has a large number of systems to acquire this essential nutrient and many of these systems have been characterised in the laboratory. However, which iron acquisition systems are active in CF is not well understood. Here we review recent research that sheds light on how P. aeruginosa obtains iron in the lungs of CF patients.     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

Nucleotide sequence and functional properties of a sodium-dependent citrate transport system from Klebsiella pneumoniae.

The gene of the sodium-dependent citrate transport system from Klebsiella pneumoniae (citS) is located on plasmid pES3 (Schwarz, E., and Oesterhelt, D. (1985) EMBO J. 4, 1599-1603) and encodes a 446-amino acid protein. Transport of citrate via this citrate transport protein (CitS) is dependent on the presence of sodium ions and is inhibited by magnesium ions. The delta pH (pH gradient across the membrane) is the major driving force for uptake. It is postulated that, in analogy with the proton-dependent citrate carrier (CitH) of K. pneumoniae (van der Rest, M. E., Abee, T., Molenaar, D., and Konings, W. N. (1990) Eur. J. Biochem. 195, 71-77), only one of the protonated species of citrate is recognized by CitS and that citrate is translocated across the membrane in symport with protons and sodium ions. The hydrophobicity profile of CitS suggests that the protein is very hydrophobic and contains 12 membrane-spanning segments. These segments are not centered around a hydrophilic core as has been suggested for other transport proteins, but the protein is asymmetrical with seven transmembrane segments in front of a large hydrophilic loop and five after this loop. The amino acid sequence is highly similar to a citrate transport system of Lactococcus lactis subsp. lactis var. diacetylactis (CitP) (David, S., van der Rest, M. E., Driessen, A. J. M., Simons, G., and de Vos, W. M. (1990) J. Bacteriol. 172, 5789-5794) and less similar to CitH of K. pneumoniae. We conclude that the citS gene of K. pneumoniae encodes a sodium-dependent citrate transport system that belongs to a novel subclass of transport proteins.     

Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells

The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.     

Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: Multiple pathways to induce tolerance?

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44 degrees C) or pretreatment with sodium-arsenite (1 hour at 37 degrees C, 100 microM). Inhibition of overall protein and heat shock protein (HSP) synthesis (greater than 95%) by cycloheximide (25 micrograms/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat-induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium-arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post-heat incubations of the cells at 37 degrees C. The rate of recovery was faster in heat-induced tolerant cells than in nontolerant cells. Arsenite-induced tolerant cells did not show an enhanced rate of recovery from the heat-induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post-heat recovery was enhanced in heat-induced, but not arsenite-induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.     

Processing of the lactococcal extracellular serine proteinase.

Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membrane-associated lipoprotein PrtM. To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed. This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme. The secreted form of this inactivated proteinase was the same size as the inactive secreted form of the proteinase produced in the absence of PrtM. Both inactive proteinases are larger than the active proteinase. Isolation of proteinase by washing lactococcal cells carrying the complete proteinase gene in a Ca(2+)-free buffer was prevented by the absence of prtM or the absence of a functional active site. We propose that PrtM, during or after membrane translocation of the proteinase, effects the autoproteolytic removal of the N-terminal pro region of the proteinase. Subsequent C-terminal autodigestion results in the release of the enzyme from the lactococcal cells.     

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.