A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis

The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.     

Coupling mechanism of the oxaloacetate decarboxylase Na(+) pump

The oxaloacetate decarboxylase Na(+) pump consists of subunits alpha, beta and gamma, and contains biotin as the prosthetic group. The peripheral alpha subunit catalyzes the carboxyltransfer from oxaloacetate to the prosthetic biotin group to yield the carboxybiotin enzyme. Subsequently, this is decarboxylated in a Na(+)-dependent reaction by the membrane-bound beta subunit. The decarboxylation is coupled to Na(+) translocation from the cytoplasm into the periplasm, and consumes a periplasmically derived proton. The gamma subunit contains a Zn(2+) metal ion which may be involved in the carboxyltransfer reaction. It is proposed to insert with its N-terminal alpha-helix into the membrane and to form a complex with the alpha subunit with its water-soluble C-terminal domain. The beta subunit consists of nine transmembrane alpha-helices, a segment (IIIa) which inserts from the periplasm into the membrane but does not penetrate it, and connecting hydrophilic loops. The most highly conserved regions of the molecule are segment IIIa and transmembrane helix VIII. Functionally important residues are D203 (segment IIIa), Y229 (helix IV) and N373, G377, S382 and R389 (helix VIII). The polar of these amino acids may constitute a network of ionizable groups which promotes the translocation of Na(+) and the oppositely oriented translocation of H(+) across the membrane. Evidence indicates that two Na(+) ions are bound simultaneously to subunit beta with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin possibly with the consumption of the proton extracted from S382 and delivered via Y229 to the carboxylated prosthetic group. A conformational change exposes the bound Na(+) ions toward the periplasm. With H(+) entering from the periplasm, the hydroxyl group of S382 is regenerated, and as a consequence, the Na(+) ions are released into this compartment. After switching back to the original conformation, Na(+) pumping continues.     

Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na(+) pump

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.     

Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.

The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.     

Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.     

Aspartate 203 of the oxaloacetate decarboxylase beta-subunit catalyses both the chemical and vectorial reaction of the Na+ pump.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.     

Site-directed sulfhydryl labeling of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae: helix VIII comprises a portion of the sodium ion channel

Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.     

Role of the intracellular domain of the beta subunit in Na,K pump function

The catalytic alpha subunit of the (Na,K)- and (H,K)-ATPases needs to be coexpressed with a beta subunit in order to produce cation transport activity. Although the isoform of the beta subunit is known to influence the functional characteristics of the Na,K pump, the role of the different domains of the beta subunit is not fully understood. We have studied the function of a Na,K pump resulting from the expression of a wild-type alpha subunit with a N-terminally truncated mutant of the beta subunit using the two-electrode voltage clamp and the cut-open oocyte techniques. While the maximal activity, measured as the K+-activated outward current, was not significantly altered, the beta N-terminal truncation induced an ouabain-sensitive conductance in the absence of extracellular K+. The voltage dependence of the ouabain-sensitive charge distribution indicated that in the Na/Na exchange conditions, the E1-E2 conformation equilibrium was shifted towards the E2 conformation, a change resulting from alteration of both the forward and the backward reaction rate. Removal of the intracellular domain of the beta subunit modifies several aspects of the whole enzyme function by a mechanism that must imply the state of the extracellular and/or transmembrane parts of the alpha/beta subunit complex.     

Identification of a domain in the alpha-subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the gamma-subunit

The oxaloacetate decarboxylase Na+ pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral alpha-subunit associated with two integral membrane-bound subunits (beta and gamma). The alpha-subunit contains the carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The gamma-subunit plays a profound role in the assembly of the complex. It interacts with the beta-subunit through its N-terminal membrane-spanning region and with the alpha-subunit through its hydrophilic C-terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD-2 expression clones for subunit alpha-2 and the C-terminal domain of gamma-2, termed gamma'-2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of alpha-2 was identified by deletion mutagenesis to be essential for the binding with gamma'-2. This portion of the alpha-subunit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin-binding domain at its C terminus. Results of site-directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with gamma'-2. This peptide therefore represents a newly identified, separate domain of the alpha-subunit and has been called the 'association domain'.     

Sequence of the sodium ion pump oxaloacetate decarboxylase from Salmonella typhimurium.

A genomic library of Salmonella typhimurium DNA was constructed in the lambda-phage EMBL3 and screened by immunoblotting for expression of the oxaloacetate decarboxylase alpha-subunit. After subcloning on plasmids the entire sequence of the oxaloacetate decarboxylase was determined. The genes encoding subunits gamma (oadG), alpha (oadA), and beta (oadB) of the decarboxylase are clustered on the chromosome in that order. A typical consensus sequence of a promoter is not found upstream of the oadG gene, but putative ribosome binding regions can be identified before each subunit gene. The amino acid sequences are highly homologous to those of oxaloacetate decarboxylase from Klebsiella pneumoniae with 71% identity between the gamma-subunits, 92% identity between the alpha-subunits, and 93% identity between the beta-subunits. The homology between the corresponding beta-subunits appeared to exist only between the 312 N-terminal amino acid residues. It was shown that a cloning artifact has occurred during DNA sequence determination of the beta-subunit from K. pneumoniae and has led to erroneous results. The sequence of this polypeptide is corrected in the Appendix to this paper. A plasmid encoding the three oad genes and that for the anaerobic citrate carrier (citS) was cloned from the chromosomal DNA and used for sequence determination.     

FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.     

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na+/H+ antiporter in Bacillus subtilis

ShaA, a member of a multigene-encoded Na+/H+ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H+-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na+/H+ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.     

Respiration-driven Na+ pump and Na+ circulation in Vibrio parahaemolyticus.

Sodium circulation in Vibrio parahaemolyticus was investigated. We observed respiration-driven Na+ extrusion from cells by using a Na+ electrode. The Na+ extrusion was insensitive to a proton conductor, carbonyl cyanide m-chlorophenylhydrazone, and sensitive to a respiratory inhibitor, CN-. These results support the idea of the existence of a respiratory Na+ pump in V. parahaemolyticus. The respiration-driven Na+ extrusion was observed only under alkaline conditions.     

The roles of the Na,K-ATPase beta 1 subunit in pump sorting and epithelial integrity

In epithelial MDCK cells, the Na,K-ATPase is co-localized with adherens junctions in all stages of monolayer formation starting from initiation of cell–cell contact. The Na,K-ATPase and adherens junction proteins stay partially co-localized even after internalization due to disruption of intercellular contacts by Ca²⁺ deprivation. Similar to adherens junction proteins, the Na,K-ATPase is resistant to extraction with non-ionic detergent, suggesting pump association with the cytoskeleton. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β₁ subunit and the endogenous α₁ subunit is easily dissociated from the adherens junctions and cytoskeleton by detergent extraction. The MDCK cells in which half of the endogenous β₁ subunits in the lateral membrane are substituted by unglycosylated β₁ subunits display a slower rate of cell-to-cell contact formation and decreased ability to both spread over the surface and migrate. The lack of N-glycans in the Na,K-ATPase β₁ subunit results in an impairment of mature cell–cell junctions as detected by an increase in the paracellular permeability of the MDCK cell monolayers and by a decrease in resistance of adherens junction proteins to extraction by a non-ionic detergent. Therefore the N-glycans of the Na,K-ATPase β₁ subunit are important for retention of the pump at the sites of cell–cell contact. Moreover, they are important for the integrity and stability of cell–cell junctions in mature epithelia. In addition, N-glycans contribute to the formation of cell–cell contacts between surface-attached dispersed cells by mediating lamellipodia formation and stabilizing the newly formed adherens junctions.     

Role of the conserved cysteine residues of the 11.5 kDa subunit in complex I catalytic properties

Mitochondrial complex I exists as a mixture of two inter-convertible forms: active (A) and de-activated (D), the latter being sensitive to SH-modifying compounds. To investigate if the conserved cysteine-rich 11.5 kDa subunit of Neurospora crassa complex I is involved in this process, we subjected the corresponding genomic DNA to site-directed mutagenesis. The four cysteine residues of the subunit were separately substituted with serine residues and the resulting proteins were independently expressed in a null-mutant strain. All of the obtained mutant strains were able to assemble a complex I with similar kinetic properties to those observed in the wild-type enzyme, indicating that none of the cysteine residues of the 11.5 kDa protein is individually relevant for the A/D transition process. Diminished amounts of assembled complex I seem to be the major effect of these specific mutations. The cysteine residues are likely important to the acquisition and stabilization of the correct 11.5 kDa protein conformation and this is reflected in the assembly/stability of complex I.     

Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane

Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.     

Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.     

The sodium ion translocating oxaloacetate decarboxylase of Klebsiella pneumoniae. Sequence of the integral membrane-bound subunits beta and gamma

The sequences upstream and downstream of the cloned gene for the alpha-subunit of the Na+ pump oxaloacetate decarboxylase of Klebsiella pneumonia were determined. An open reading frame in the upstream region was identified as the gene for the gamma-subunit, and an open reading frame in the downstream region represents the gene for the beta-subunit. The deduced primary structure of the gamma- and beta-subunit was confirmed by protein sequencing of about 37 and 22%, respectively, of each polypeptide chain. The gene for the gamma-subunit has a GC content of 64% and codes for 83 amino acids. The protein is not processed at its amino terminus or at its carboxyl terminus. The gene for the beta-subunit has a GC content of 66% and codes for 327 amino acids. The protein contains a blocked aminoterminal methionine residue. Whether processing occurs at the carboxyl terminus is unknown. Hydropathy calculations defined one transmembrane helix in the amino-terminal part of the gamma-subunit and a hydrophilic carboxyl-terminal part that is certainly not embedded within the lipid bilayer. A proline- and alanine-rich sequence in the carboxyl-terminal part may provide the protein with conformational flexibility. According to hydropathy and acrophilicity calculations, the secondary structure of the beta-subunit may be formed with 5 or 6 intramembrane helical segments.     

Na+ as coupling ion in energy transduction in extremophilic Bacteria and Archaea

For microorganisms to live under extreme physical conditions requires important adaptations of the cells. In many organisms the use of Na⁺ instead of protons as coupling ion in energy transduction is associated with such adaptation. This review focuses on the enzymes that are responsible for the generation and utilization of Na⁺ gradients in extremophilic microorganisms. Aspects that are dealt with include: bioenergetics and ion homeostasis in extremophilic Bacteria and Archaea; the molecular mechanism of Na⁺ translocation; and (dis)advantages of Na⁺ as coupling ion in energy transduction.G. Speelmans was and B. Poolman and W.N. Konings are with the Department of Microbiology, Biology Centre University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands; G. Speelmans is now with the Department of Biochemistry of Membranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.