The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.     

The YedZ family: possible heme binding proteins that can be fused to transporters and electron carriers

YedZ of Escherichia coli is an integral 6 transmembrane spanning (TMS) protein of unknown function. We have identified homologues of YedZ in bacteria and animals but could not find homologues in Archaea or the other eukaryotic kingdoms. YedZ homologues exhibit conserved histidyl residues in their transmembrane domains that may function in heme binding. Some of the homologues encoded in the genomes of magnetotactic bacteria and cyanobacteria have YedZ domains fused to transport and electron transfer proteins, respectively. One of the animal homologues is the 6 TMS epithelial plasma membrane antigen of the prostate (STAMP1) that is overexpressed in prostate cancer. Animal homologues have YedZ domains fused C-terminal to homologues of coenzyme F420-dependent NADP oxidoreductases. YedZ homologues are shown to have arisen by intragenic triplication of a 2 TMS-encoding element. They exhibit slight but statistically significant sequence similarity to two families of putative heme export systems and one family of cytochrome-containing electron carriers. We propose that YedZ homologues function as heme-binding proteins that can facilitate or regulate oxidoreduction, transmembrane electron flow and transport.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

Polar residues in a conserved motif spanning helices 1 and 2 are functionally important in the SulP transporter family

The SulP family (including the SLC26 family) is a diverse family of anion transporters found in all domains of life, with different members transporting different anions. We used sequence and bioinformatics analysis of helices 1 and 2 of SulP family members to identify a conserved motif, extending the previously defined 'sulfate transporter motif'. The analysis showed that in addition to being highly conserved in both sequence and spacing, helices 1 and 2 contain a significant number of polar residues and are predicted to be buried within the protein interior, with at least some faces packed closely against other helices. This suggests a significant functional role for this region and we tested this by mutating polar residues in helices 1 and 2 in the sulfate transporter, SHST1. All mutations made, even those removing only a single hydroxyl group, had significant effects on transport. Many mutations abolished transport without affecting plasma membrane expression of the mutant protein, suggesting a functional role for these residues. Different helical faces appear to have different roles, with the most severe effects being localised to two interacting faces of helices 1 and 2. Our results confirm the predicted importance of conserved polar residues in helices 1 and 2 and suggest that transport of sulfate by SHST1 is dependent on a network of polar and aromatic interactions between these two helices.     

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.     

A novel ubiquitous family of putative efflux transporters

We describe a novel family of putative efflux transporters (PET) found in bacteria, yeast and plants. None of the members of the PET family has been functionally characterized. The bacterial and yeast proteins display a duplicated internal repeat element consisting of an N-terminal hydrophobic sequence of about 170 residues, exhibiting six putative transmembrane alpha-helical spanners (TMSs), followed by a large (230 residue), C-terminal, hydrophilic, cytoplasmic domain. The plant proteins exhibit only one such unit, but they have a larger C-terminal cytoplasmic domain. Arabidopsis thaliana encodes at least seven paralogues of the PET family. The gram-negative bacterial proteins are sometimes encoded by genes that are found in operons that also contain genes that encode membrane fusion proteins. This fact strongly suggests that PET family proteins are efflux pumps. The sequence, topological and phylogenetic characteristics of these proteins as well as the operonic structures of their encoded genes when relevant are described.     

The complete phosphotransferase system in Escherichia coli

We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively. In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.     

Survey for g-proteins in the prokaryotic genomes: prediction of functional roles based on classification

The members of the family of G-proteins are characterized by their ability to bind and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). Despite a common biochemical function of GTP hydrolysis shared among the members of the family of G-proteins, they are associated with diverse biological roles. The current work describes the identification and detailed analysis of the putative G-proteins encoded in the completely sequenced prokaryotic genomes. Inferences on the biological roles of these G-proteins have been obtained by their classification into known functional subfamilies. We have identified 497 G-proteins in 42 genomes. Seven small GTP-binding protein homologues have been identified in prokaryotes with at least two of the diagnostic sequence motifs of G-proteins conserved. The translation factors have the largest representation (234 sequences) and are found to be ubiquitous, which is consistent with their critical role in protein synthesis. The GTP_OBG subfamily comprises of 79 sequences in our dataset. A total of 177 sequences belong to the subfamily of GTPase of unknown function and 154 of these could be associated with domains of known functions such as cell cycle regulation and t-RNA modification. The large GTP-binding proteins and the alpha-subunit of heterotrimeric G-proteins are not detected in the genomes of the prokaryotes surveyed.     

NMR assignment and secondary structure of the STAS domain of Rv1739c,a putative sulfate transporter of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

We report ¹H^N, ¹⁵N, and ¹³C resonance assignments for the 15.6 kDa STAS domain of the putative sulfate transporter of Mycobacterium tuberculosis, Rv1739c, using heteronuclear, multidimensional NMR spectroscopy. Rv1739c is a SulP anion permease, related in structure to the SLC26 gene family of metazoan anion exchangers and anion channels.     

Defense Against Cannibalism: The SdpI Family of Bacterial Immunity/Signal Transduction Proteins

The SdpI family consists of putative bacterial toxin immunity and signal transduction proteins. One member of the family in Bacillus subtilis, SdpI, provides immunity to cells from cannibalism in times of nutrient limitation. SdpI family members are transmembrane proteins with 3, 4, 5, 6, 7, 8, or 12 putative transmembrane α-helical segments (TMSs). These varied topologies appear to be genuine rather than artifacts due to sequencing or annotation errors. The basic and most frequently occurring element of the SdpI family has 6 TMSs. Homologues of all topological types were aligned to determine the homologous TMSs and loop regions, and the positive-inside rule was used to determine sidedness. The two most conserved motifs were identified between TMSs 1 and 2 and TMSs 4 and 5 of the 6 TMS proteins. These showed significant sequence similarity, leading us to suggest that the primordial precursor of these proteins was a 3 TMS–encoding genetic element that underwent intragenic duplication. Various deletional and fusional events, as well as intragenic duplications and inversions, may have yielded SdpI homologues with topologies of varying numbers and positions of TMSs. We propose a specific evolutionary pathway that could have given rise to these distantly related bacterial immunity proteins. We further show that genes encoding SdpI homologues often appear in operons with genes for homologues of SdpR, SdpI’s autorepressor. Our analyses allow us to propose structure–function relationships that may be applicable to most family members.     

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

Sodium and sulfate ion transport in yeast vacuoles

The intra-luminal acidic pH of endomembrane organelles is established by a proton pump, vacuolar H(+)-ATPase (V-ATPase), in combination with other ion transporter(s). The proton gradient (DeltapH) established in yeast vacuolar vesicles decreased and reached the lower value after the addition of alkaline cations including Na(+). As expected, the uptake of (22)Na(+) was coupled with DeltapH generated by V-ATPase. Disruption of NHX1 or NHA1, encoding known Na(+)/H(+) antiporters, did not result in the loss of (22)Na(+) uptake or the alkaline cation-dependent DeltapH decrease. Upon the addition of sulfate ions, the V-ATPase-dependent DeltapH in the vacuolar vesicles increased, but the membrane potential (DeltaPsi) decreased. Consistent with this observation, radioactive sulfate was transported into the vesicles with a K(m) value of 0.07 mM. The transport activity was unaffected upon disruption of the putative genes coding for homologues of plasma membrane sulfate transporters. These results indicate that the vacuoles exhibit unique Na(+)/H(+) antiport and sulfate transport, which regulate the luminal pH and ion homeostasis in yeast.     

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.