Membrane topology of the cyanobacterial bicarbonate transporter,BicA, a member of the SulP (SLC26A) family

Abstract

We have completed the first comprehensive transmembrane topology determination for a member of the ubiquitous and important SulP/SLC26 family of coupled anion transporters found in eukaryotes and prokaryotes. The prokaryotic member that we have mapped, namely BicA from Synechococcus PCC7002, is an important Na⁺-dependent bicarbonate transporter that is likely to play a major role in global primary productivity via the CO₂ concentrating mechanism in cyanobacteria. We experimentally determined the topology based on phoA-lacZ topology mapping combined with reference to a range of predictive models based on hydropathy analysis and positive charge distribution. The 12-TMH structure for BicA is characterized by tight turns between several pairs of TMH and it features a prominent cytoplasmically-located STAS domain that is characteristic of the SulP family. A key difference from previous predicted models is that we identify a cytoplasmic loop between helices 8 and 9 where previous models suggested a TMH. This region includes a highly conserved motif that defines the SulP family. The identification of this region as cytoplasmic, rather than transmembrane, has implications for the function and perhaps regulation of SulP family members. This finding is used to reinterpret mutagenesis data relating to highly conserved residues in this region from both plant and human SulP transporters.     

Membrane topology of the cyanobacterial bicarbonate transporter,SbtA, and identification of potential regulatory loops

Abstract

The transporter SbtA is a high affinity Na⁺-dependent HCO₃ ^- uptake system present in a majority of cyanobacterial clades. It functions in conjunction with CO₂ uptake systems and other HCO₃ ^- uptake systems to allow cyanobacteria to accumulate high levels of HCO₃ ^- used to support efficient photosynthetic CO₂ fixation via the CO₂ concentrating mechanism in these species. The phoA/lacZ fusion reporter method was used to determine the membrane topology of the cyanobacterial bicarbonate transporter, SbtA (predicted size of ～ 39.7 kD), cloned from the freshwater strain, Synechocystis PCC6803. The structure conforms to a model featuring 10 transmembrane helices (TMHs), with a distinct 5 + 5 duplicated structure. Both the N- and C-terminus are outside the cell and the second half of the protein is inverted relative to the first. The first putative helix appears to lack sufficient topogenic signals for its correct orientation in the membrane and instead relies on the presence of later helices. The cytoplasmic loop between helices 5 and 6 is a likely location for regulatory mechanisms that could govern activation of the transporter, and the cytoplasmic loop between helices 9 and 10 also contains some conserved putative regulatory residues.     

Membrane topology of the cyanobacterial bicarbonate transporter, SbtA, and identification of potential regulatory loops

The transporter SbtA is a high affinity Na+-dependent HCO3- uptake system present in a majority of cyanobacterial clades. It functions in conjunction with CO2 uptake systems and other HCO3- uptake systems to allow cyanobacteria to accumulate high levels of HCO3- used to support efficient photosynthetic CO2 fixation via the CO2 concentrating mechanism in these species. The phoA/lacZ fusion reporter method was used to determine the membrane topology of the cyanobacterial bicarbonate transporter, SbtA (predicted size of ～39.7 kD), cloned from the freshwater strain, Synechocystis PCC6803. The structure conforms to a model featuring 10 transmembrane helices (TMHs), with a distinct 5+5 duplicated structure. Both the N- and C-terminus are outside the cell and the second half of the protein is inverted relative to the first. The first putative helix appears to lack sufficient topogenic signals for its correct orientation in the membrane and instead relies on the presence of later helices. The cytoplasmic loop between helices 5 and 6 is a likely location for regulatory mechanisms that could govern activation of the transporter, and the cytoplasmic loop between helices 9 and 10 also contains some conserved putative regulatory residues.     

Characterisation of Cyanobacterial Bicarbonate Transporters in E. coli Shows that SbtA Homologs Are Functional in This Heterologous Expression System

Cyanobacterial HCO₃ ^- transporters BCT1, SbtA and BicA are important components of cyanobacterial CO₂-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six β-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO₃ ^- transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.     

The cyanobacterial bicarbonate transporter BicA: its physiological role and the implications of structural similarities with human SLC26 transporters

The cyanobacterial Na+-dependent HCO3- transporter BicA is a member of the ubiquitous and important SulP/SLC26 family of anion transporters found in eukaryotes and prokaryotes. BicA is an important component of the cyanobacterial CO2 concentrating mechanism, an adaptation that contributes to cyanobacteria being able to achieve an estimated 25% of global primary productivity, largely in the oceans. The human SLC26 members are involved in a range of key cellular functions involving a diverse range of anion transport activities including Cl-/HCO3-, I-/HCO3-, and SO42-/HCO3- exchange; mutations in SLC26 members are known to be associated with debilitating diseases such as Pendred syndrome, chondrodysplasias, and congenital chloride diarrhoea. We have recently experimentally determined the membrane topology of BicA using the phoA-lacZ reporter system and here consider some of the extrapolated implications for topology of the human SLC26 family and the Sultr plant sulphate transporters.     

Sequence analyses of cyanobacterial bicarbonate transporters and their homologues

The primary HCO3- uptake system in the cyanobacterium Synecocystis is the Na+-dependent transporter SbtA. SbtA and its homologues were identified and shown to display a common topology of ten transmembrane segments (TMSs). These proved to have arisen by an intragenic duplication event from an ancestral gene encoding a five TMS protein product. A region of SbtA shows sufficient similarity to 10 TMS ABC-type integral membrane transport proteins to suggest a common origin. Phylogenetic analyses of the SbtA family revealed two clusters of cyanobacterial homologues with all non-cyanobacterial family members outside of these two clusters. The tree topology suggests that SbtA family members display multiple transport functions.     

Membrane topology mapping of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis

The membrane topologies of the six subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na⁺-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.     

Characterisation and cloning of a Na-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B transporter family

Na⁺-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B⁰. This transporter is shown to differ in specificity from the B⁰ transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B⁰ transporter family we have isolated a cDNA encoding the NBL-1 cell System B⁰ transporter. When expressed in Xenopus oocytes the clone catalysed Na⁺-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na⁺-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B⁰/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B⁰ transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

Membrane topology of the p1258 CadA Cd(II)/Pb(II)/Zn(II)-translocating P-type ATPase

Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X)₂Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments.     

Membrane Topology Motifs in the SGLT Cotransporter Family

Homologues of the Na⁺/glucose cotransporter, the SGLT family, include sequences of mammalian, eubacterial, yeast, insect and nematode origin. The cotransported substrates are sugars, inositol, proline, pantothenate, iodide, urea and undetermined solutes. It is reasonable to expect that the SGLT family members share a similar or identical topology of membrane spanning elements, by virtue of their common ancestry and similar coupling of solute transport to downhill sodium flux. Here we examine their membrane topologies as deduced from diverse analyses of their primary sequences, and from their sequence correlations with the experimentally determined topology of the human Na⁺/glucose cotransporter SGLT1. Our analyses indicate that all family members share a common core of 13 transmembrane helices, but that some, like SGLT1 itself, have one additional span appended to the C-terminus, and still others, two. One bacterial member incorporates an additional span at the N-terminus. Sequence comparisons indicative of common ancestry of the SGLT and the [Na⁺+ Cl⁻] transporter families are introduced, and evaluated in light of their topologies. New evidence concerning the previously asserted common ancestry of SGLT1 and an N-acetylglucosamine permease of the bacterial phosphotransferase system is considered. Finally, we analyze observations which lead us to conjecture that the experimental strategy most commonly employed to reveal the topology of bacterial transporters (i.e., the fusion of reporter enzymes such as phoA alkaline phosphatase, beta-lactamase or beta-galactosidase, to progressively C-truncated fragments of the transporter) has often instead so perturbed local topology as to have entirely missed pairs of adjacent membrane spans. Received: 18 May 1996     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Installing extra bicarbonate transporters in the cyanobacterium Synechocystis sp. PCC6803 enhances biomass production

As a means to improve carbon uptake in the cyanobacterium Synechocystis sp. strain PCC6803, we engineered strains to contain additional inducible copies of the endogenous bicarbonate transporter BicA, an essential component of the CO₂-concentrating mechanism in cyanobacteria. When cultured under atmospheric CO₂ pressure, the strain expressing extra BicA transporters (BicA⁺ strain) grew almost twice as fast and accumulated almost twice as much biomass as the control strain. When enriched with 0.5% or 5% CO₂, the BicA⁺ strain grew slower than the control but still showed a superior biomass production. Introducing a point mutation in the large C-terminal cytosolic domain of the inserted BicA, at a site implicated in allosteric regulation of transport activity, resulted in a strain (BicA⁺_(T485G) strain) that exhibited pronounced cell aggregation and failed to grow at 5% CO₂. However, the bicarbonate uptake capacity of the induced BicA⁺_(T485G) was twice higher than for the wild-type strain. Metabolic analyses, including phenotyping by synchrotron-radiation Fourier transform Infrared spectromicroscopy, scanning electron microscopy, and lectin staining, suggest that the excess assimilated carbon in BicA⁺ and BicA⁺_(T485G) cells was directed into production of saccharide-rich exopolymeric substances. We propose that the increased capacity for CO₂ uptake in the BicA⁺ strain can be capitalized on by re-directing carbon flux from exopolymeric substances to other end products such as fuels or high-value chemicals.     

How pH regulates a pH regulator: a regulatory hot spot in the N-terminal cytoplasmic domain of the AE2 anion exchanger

Regulation of cell pH and cell volume require homeostatic control of intracellular cations and anions. Bicarbonate transporters play an important role in these cellular functions. The SLC4 and SLC26 gene families both encode bicarbonate transporter polypeptides. The SLC4 gene family includes four Na⁺-independent chloride-bicarbonate exchanger genes and multiple Na⁺-bicarbonate cotransporter and Na⁺-dependent anion-exchanger genes. The acute regulatory properties of the recombinant polypeptides encoded by these genes remain little studied. The most extensively studied among them are the Na⁺-independent anion exchangers AE1, AE2, and AE3. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by the ammonium ion. These properties are not shared by the closely related AE1 anion exchanger of the erythrocyte and the renal collecting duct Type A intercalated cell. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2, which are important in the exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.     

Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis

The transmembrane topology of the Acr3 family arsenite transporter Acr3 from Bacillus subtilis was analysed experimentally using translational fusions with alkaline phosphatase and green fluorescent protein and in silico by topology modelling. Initial topology prediction resulted in two models with 9 and 10 TM helices respectively. 32 fusion constructs were made between truncated forms of acr3 and the reporter genes at 17 different sites throughout the acr3 sequence to discriminate between these models. Nine strong reporter protein signals provided information about the majority of the locations of the cytoplasmic and extracellular loops of Acr3 and showed that both the N- and the C-termini are located in the cytoplasm. Two ambiguous data points indicated the possibility of an alternative 8 helix topology. This possibility was investigated using another 10 fusion variants, but no experimental support for the 8 TM topology was obtained. We therefore conclude that Acr3 has 10 transmembrane helices. Overall, the loops which connect the membrane spanning segments are short, with cytoplasmic loops being somewhat longer than the extracellular loops. The study provides the first ever experimentally derived structural information on a protein of the Acr3 family which constitutes one of the largest classes of arsenite transporters.     

Carbohydrate uptake genes inEscherichia coli are induced by carbon starvation

Escherichia coli produces several membrane-associated and periplasmic proteins in response to deprivation for a carbon source. The carbon starvation response involves a two- to fourfold, cAMP-dependent induction of operons involved in carbohydrate uptake and utilization, including thelac operon. Threecarbon-starvation-inducible (sci) gene fusions to aphoA reporter sequence were characterized. ThephoA-fusions werecya ⁺/crp ⁺-dependent and located in three previously characterized genes involved in high-affinity uptake of alternative carbon sources:mglB, encoding the periplasmic galactose binding protein;rbsB, encoding the periplasmic ribose binding protein; andlamB, encoding the maltodextrin-specific outer membrane porin.     

Role of habitat and great oxidation event on the occurrence of three multisubunit inorganic carbon-uptake systems in cyanobacteria

The oxygenase reaction catalyzed by RuBisCO became an issue only after the evolution of the oxygenic photosynthesis in cyanobacteria. Several strategies were developed by autotrophic organisms as an evolutionary response to increase oxygen levels to help RuBisCO maximize its net carboxylation rate. One of the crucial advancements in this context was the development of more efficient inorganic carbon transporters which could help in increasing the influx of inorganic carbon (Ci) at the site of CO₂ fixation. We conducted a survey to find out the genes coding for cyanobacterial Ci transporters in 40 cyanobacterial phyla with respect to transporters present in Gloeobacter violaceous PCC 7421, an early-diverging cyanobacterium. An attempt was also made to correlate the prevalence of the kind of transporter present in the species with its habitat. Basically, two types of cyanobacterial inorganic carbon transporters exist, i.e. bicarbonate transporters and CO₂-uptake systems. The transporters also show variation in context to their structure as some exist as single subunit proteins (BicA and SbtA), while others exist as multisubunit proteins (namely BCT1, NdhI₃ and NdhI₄). The phylogeny and distribution of the former have been extensively studied and the present analysis provides an insight into the latter ones. The in silico analysis of the genes under study revealed that their distribution was greatly influenced by the habitat and major environmental changes such as the great oxidation event (GOE) in the course of their evolution.     

Model-Guided Mutagenesis Drives Functional Studies of Human NHA2, Implicated in Hypertension

Human NHA2 is a poorly characterized Na⁺/H⁺ antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.