HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 27, 20551-20555, July 7, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20551    most recent M003034200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maeda, S.-i. Articles by Omata, T. Search for Related Content PubMed PubMed Citation Articles by Maeda, S.-i. Articles by Omata, T. Social Bookmarking                      What's this?

Bicarbonate Binding Activity of the CmpA Protein of the Cyanobacterium Synechococcus sp. strain PCC 7942 Involved in Active Transport of Bicarbonate^*

Shin-ichi Maeda^§, G. Dean Price^§, Murray R. Badger^§, Chika Enomoto^¶, and Tatsuo Omata^¶

From the  Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra ACT 2601, Australia and the ^¶ Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Received for publication, April 11, 2000, and in revised form, April 21, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cmpABCD operon of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an ATP-binding cassette transporter involved in HCO₃ uptake. The three genes, cmpBCD, encode membrane components of an ATP-binding cassette transporter, whereas cmpA encodes a 42-kDa cytoplasmic membrane protein, which is 46.5% identical to the membrane-anchored substrate-binding protein of the nitrate/nitrite transporter. Equilibrium dialysis analysis using H¹⁴CO₃ showed that a truncated CmpA protein lacking the N-terminal 31 amino acids, expressed in Escherichia coli cells as a histidine-tagged soluble protein, specifically binds inorganic carbon (CO₂ or HCO₃). The addition of the recombinant CmpA protein to a buffer caused a decrease in the concentration of dissolved CO₂ because of the binding of inorganic carbon to the protein. The decrease in CO₂ concentration was accelerated by the addition of carbonic anhydrase, indicating that HCO₃, but not CO₂, binds to the protein. Mass spectrometric measurements of the amounts of unbound and bound HCO₃ in CmpA solutions containing low concentrations of inorganic carbon revealed that CmpA binds HCO₃ with high affinity (K_d = 5 µM). A similar dissociation constant was obtained by analysis of the competitive inhibition of the CmpA protein on the carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxylase at limiting concentrations of HCO₃. These findings showed that the cmpA gene encodes the substrate-binding protein of the HCO₃ transporter.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyanobacteria possess multiple transporters for uptake of inorganic carbon (CO₂ and HCO₃; designated C_i)¹ into the cell. The cells accumulate C_i in the cytoplasm as HCO₃ and convert it into CO₂ in carboxysomes, the polyhedral inclusion bodies to which ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is localized, to elevate the CO₂ concentration around the CO₂-fixing enzyme (1, 2). The transport of inorganic carbon has been the least understood step of carbon assimilation in cyanobacteria. We have, however, recently identified a high affinity HCO₃ transporter (BCT1) of Synechococcus sp. strain PCC 7942 (3). The transporter is encoded by the four genes cmpA, cmpB, cmpC, and cmpD (4, 5), which form a low CO₂-inducible operon (3). The cmpA, cmpB, cmpC, and cmpD genes are strongly similar to the genes encoding the nitrate/nitrite transporter, nrtA, nrtB, nrtC, and nrtD, respectively, of the same organism (6-8). Similar to nrtB, cmpB encodes a hydrophobic protein with structural similarities to the integral membrane components of ABC transporters; cmpC and cmpD encode the ATP-binding cassette proteins strongly similar to nrtC and nrtD, respectively. The product of cmpA is a 42-kDa cytoplasmic membrane protein, which is 46.5% identical to the nrtA gene product that functions as the membrane-anchored substrate (nitrate and nitrite)-binding protein (9). The similarity of CmpA to NrtA and its involvement in HCO₃ uptake strongly suggest that the product of cmpA is the substrate-binding protein of the HCO₃ transporter. In this work, we have biochemically verified this assumption by showing high affinity binding of HCO₃ to a recombinant CmpA protein. Although contamination by atmospheric CO₂ made it impossible to determine the dissociation constant of CmpA and HCO₃ by the equilibrium dialysis technique using H¹⁴CO₃, use of a mass spectrometer enabled measurements of the amounts of free and bound H¹²CO₃ in CmpA solutions containing low concentrations of H¹²CO₃, allowing kinetic analysis of binding of HCO₃ to the protein. The dissociation constant was estimated also from the competitive inhibition of CmpA on the phosphoenolpyruvate carboxylase (PEPCase) reaction in the presence of limiting amounts of HCO₃. Both methods resulted in dissociation constant of 5 µM for CmpA and HCO₃, demonstrating that the cmpA gene encodes a HCO₃-binding protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Recombinant CmpA-- A 1.3-kilobase pair DNA fragment, carrying a truncated cmpA coding region lacking the first 93 bases, was cloned between the BamHI and SmaI sites in the polylinker of the expression vector pQE-32 (QIAGEN). The resulting plasmid (pQECMPA) carried a chimeric gene, which encodes a fusion protein consisting of an N-terminal amino acid segment carrying six consecutive histidine residues (MRGSH₆GI) and truncated CmpA lacking the N-terminal 31 amino acids. The plasmid was transformed into Escherichia coli M15 (pREP4) (QIAGEN), expression of the chimeric gene was induced by 1 mM isopropyl-1-thio--D-galactopyranoside (IPTG), and the histidine-tagged protein was purified on Ni²⁺- (or Co²⁺-) nitrilotriacetic acid resin (10). The purified protein solution was dialyzed for 18 h at 15 °C against a buffer containing 20 mM TES-NaOH, pH 7.5, and 100 mM NaCl, with continuous sparging with N₂ gas.

Assay of C_i Binding Using H¹⁴CO₃-- Analysis by equilibrium dialysis, using H¹⁴CO₃, of the binding of C_i to the recombinant CmpA protein was performed at 30 °C in a buffer containing 20 mM sodium phosphate, pH 8.0, and 100 mM NaCl; 1.36-ml aliquots of protein solution (8.0 mg/ml) were dialyzed for 2 h against the same volume of the buffer supplemented with 100 µM H¹⁴CO₃ (1 × 10⁵ dpm), using paired Teflon cells separated by dialysis membrane (Spectrum). The radioactivity of ¹⁴C in each of the cells was determined with a scintillation counter (LSC-5100; Aloka).

Assay of C_i Binding at Low C_i Concentrations-- Aliquots of protein solution (10.9 mg/ml) were placed in dialysis tubes and dialyzed separately against various volumes of a buffer containing 20 mM TES-NaOH, pH 7.0 or 7.5, and 100 mM NaCl for 18 h at 30 °C. To lower the C_i concentration in buffer, the buffer was sparged continuously with N₂ gas during the dialysis, with each beaker partially sealed within a plastic "glove." Different final C_i concentrations were obtained by varying the rate of sparging with N₂ and the volume of the dialysis buffer. Using gas-tight syringes, known volumes of the protein solution and the dialysis buffer were withdrawn from the dialysis system and immediately injected into the stoppered cuvette for determination of total ¹²C_i with a mass spectrometer (see below). The concentration of the bound C_i was calculated by subtraction of total C_i concentration in the dialysis buffer from that in the protein solution. The concentration of free HCO₃ was calculated from the total C_i concentration in the dialysis buffer and the distribution between CO₂ and HCO₃ at the pH of the dialysis buffer (see below).

Mass Spectrometric Measurements of CO₂ and total C_i-- The CO₂ concentration in the aqueous solution was measured by the use of a thermostatted glass cuvette connected to a mass spectrometer (VG Micromass 6), as described previously (11, 12). The mass spectrometer was calibrated for CO₂ concentration by injection of 2 µl of 100 mM NaHCO₃ solution into 4 ml of 0.2 N HCl in the stoppered cuvette. This calibration was then used to calculate the distribution between CO₂ and HCO₃ in the cuvette at the pH of assay buffer by injection of 20 µl of 100 mM NaHCO₃ into the cuvette and the measurement of the resulting CO₂ concentration. The total C_i concentrations in the sample solutions were measured by injection of known volumes of the solutions into 0.2 N HCl and measuring the resulting CO₂ concentration.

Determination of the C_i Species That Binds to CmpA-- The C_i species that binds to CmpA was determined by examination of the disequilibrium of CO₂ and HCO₃, caused by the addition of free CmpA into buffer containing low concentrations of the C_i species. The protein solution used, containing 19.6 mg/ml (equal to 400 µM) of the recombinant CmpA protein and 0.040 µM free CO₂ (72 µM total C_i), was prepared by dialysis of a protein solution for 18 h at 15 °C against a buffer containing 20 mM TES-NaOH, pH 7.5, and 100 mM NaCl, with continuous sparging with N₂ gas as described above. A 0.5-ml aliquot of the protein solution was injected into 2.5 ml of the buffer containing 6 µM CO₂ (170 µM total C_i) with or without 1800 units/ml of carbonic anhydrase (CA) (Sigma), which had been placed in the glass cuvette thermostatted at 15 °C. The decrease in CO₂ concentration in the solution was followed with a mass spectrometer. In control experiments, the dialysis buffer equilibrated with the protein solution, containing 0.040 µM CO₂ (1.1 µM total C_i), was injected in place of the protein solution.

PEPCase Assay-- Dependence of PEPCase activity on HCO₃ concentration in assay buffer was determined in the presence and absence of the recombinant CmpA protein, which was prepared by dialysis for 18 h at 4 °C against a buffer containing 50 mM TES-NaOH, pH 7.5, and 20 mM MgCl₂, with continuous sparging of the buffer with N₂ gas. PEPCase activity was assayed at 30 °C by spectrophotometrically measuring consumption of NADH due to the coupled reduction of oxaloacetate to malate, catalyzed by malate dehydrogenase (13). Following a 3-min preincubation of the PEPCase in a 1.32-ml assay mixture containing 50 mM TES-NaOH, pH 7.5, 5 mM MgCl₂, 2 mM dithiothreitol, 0.8 mM NADH, 0.5 mM NaHCO₃, 0.17 units of PEPCase from maize (gift from Dr. Hal Hatch, CSIRO, Canberra), and 90 units of malate dehydrogenase from pig heart (Roche Molecular Biochemicals), with or without 4.2 mg of the CmpA protein, the reaction was initiated by the addition of 84 µl of 100 mM phosphoenolpyruvate. The rate of reaction was calculated from the slope of the time course of the decrease in NADH and plotted against the HCO₃ concentration at that time point. The HCO₃ concentration was calculated from the difference between the NADH concentration at the time point and that after cessation of the reaction due to complete consumption of HCO₃, using the molar ratio of 1:1 for the amounts of HCO₃ and NADH consumed in the reaction.

Protein Analyses-- Extracts of E. coli and protein samples were suspended in the sample buffer for SDS-polyacrylamide gel electrophoresis (14) and lysed by heat treatment at 100 °C for 5 min. After gel electrophoresis in the buffer system of Laemmli (14), polypeptides were stained with Coomassie Brilliant Blue. Protein concentration was determined with Coomassie Plus Protein Assay Reagent (Pierce).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of a Recombinant CmpA Protein-- The deduced CmpA polypeptide has a presumed lipoprotein signal peptide (amino acids 1-28). Truncated CmpA (amino acids 32-450) lacking the presumed signal peptide was expressed as a histidine-tagged protein in E. coli. The expression of a 44-kDa protein was induced by IPTG (Fig. 1, lanes 1 and 2). The 44-kDa protein was collected in the soluble fraction (Fig. 1, lane 3) and was purified to near homogeneity by Ni²⁺-nitrilotriacetic acid resin (Fig. 1, lane 4) or Co²⁺ nitrilotriacetic acid resin (data not shown).

View larger version (125K): [in this window] [in a new window]   Fig. 1.   Expression in E. coli and purification of the recombinant CmpA protein. Truncated CmpA (amino acids 32-450) lacking the presumed lipoprotein signal peptide was expressed as a histidine-tagged protein in E. coli and purified on Ni2+-nitrilotriacetic acid resin. Proteins were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, total protein from the E. coli expression strain before IPTG treatment; lane 2, total protein from the expression strain after 2-h treatment with IPTG; lane 3, soluble fraction from the IPTG-induced expression strain; lane 4, the protein purified on Ni2+-nitrilotriacetic acid resin; M, molecular mass markers (masses are indicated in kilodaltons). The amounts of the loaded protein were 30 µg in lanes 1-3 and 5 µg in lane 4.

Specific Binding of C_i to the Recombinant CmpA Protein-- Table I shows the distribution of ¹⁴C between 1.36-ml aliquots of the solution of the recombinant CmpA (8 mg/ml) and the dialysis buffer after equilibrium dialysis with NaH¹⁴CO₃. Under the experimental conditions, the protein solution contained 64% higher levels of ¹⁴C than the dialysis buffer. The accumulation of ¹⁴C in the protein solution was abolished by an excess amount of NaH¹²CO₃ but not with equivalent concentrations of NaNO₃, NaNO₂, Na₂SO₄, and Na₂SO₃, indicating that the truncated CmpA specifically binds C_i. In parallel experiments with no added CmpA and NaH¹⁴CO₃, however, mass spectrometric analysis showed that the solutions in the dialysis cells contain 150-200 µM of ¹²C_i due to contamination by atmospheric CO₂. Kinetic analysis of the binding of C_i to the recombinant CmpA at low C_i concentrations (<100 µM) was thus problematical by this method.

View this table: [in this window] [in a new window]   Table I Binding of inorganic carbon to recombinant CmpA protein 1.36 ml of the protein solution (8 mg/ml) was equilibrated with an equal volume of dialysis buffer containing 1 × 105 dpm (0.14 µmol) of H14CO3, and the distribution of 14Ci between the CmpA solution and the dialysis buffer was determined. The competitive substrates were added to a final concentration of 3 mM to the dialysis buffer.

The C_i Species That Binds to the CmpA Protein-- A mass spectrometer assay was employed to determine the C_i species that binds to CmpA protein. When 0.5 ml of buffer containing 0.040 µM free CO₂ (1.1 µM total free C_i) was injected into 2.5 ml of the same buffer containing 6 µM CO₂ (170 µM total C_i), concentration of the dissolved CO₂ decreased by 1.0 µM in 25 s (Fig. 2A). CA did not affect the rate of decrease in the CO₂ concentration (Fig. 2A), indicating that injection of buffer did not cause disequilibrium of the CO₂ to HCO₃ ratio and that the CO₂ decrease was due solely to C_i dilution. After injection of 0.5 ml of the protein solution (19.6 mg/ml) that had been equilibrated with the buffer containing 0.040 µM CO₂, the CO₂ concentration decreased by 2.6 µM in 400 s, verifying the ability of the recombinant CmpA protein to bind C_i. From the difference in the extent of decrease in dissolved CO₂ caused by injection of the buffer and the protein solution, the concentration of the C_i-binding site that bound C_i after the injection was calculated to be 45 µM. In the presence of CA, the extent of decrease in dissolved CO₂ was the same as that in the absence of CA, but the decrease was faster and completed in 25 s after the injection of the protein solution (Fig. 2A). These results indicated that the binding of C_i to the protein caused temporary disequilibrium of CO₂ versus HCO₃ in favor of CO₂, strongly suggesting that the CmpA protein binds HCO₃ rather than CO₂.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Disequilibrium of CO2 and HCO3 caused by binding of Ci to CmpA. A, effects of carbonic anhydrase on the changes in the CO2 concentration in a buffer after the addition of purified recombinant CmpA protein. A 0.5-ml aliquot of a CmpA solution (19.6 mg/ml), equilibrated by dialysis with a buffer containing 0.040 µM of dissolved CO2, was injected at time 0 into 2.5 ml of buffer containing 6 µM CO2 (170 µM Ci), with () or without () 1800 units of CA/ml at 15 °C. The CO2 concentration in the buffer was measured with a mass spectrometer. In control experiments, 0.5 ml of the dialysis buffer, containing 0.040 µM of dissolved CO2, was injected in place of the protein solution with () or without () CA. B, time course of decrease in CO2 concentration in the buffer caused by binding of Ci to CmpA in the presence () and absence () of CA, excluding the dilution effect, as obtained by subtraction of the curves after injection of buffer solution (circles in A) from those after injection of the protein solution (triangles in A).

The time course of decrease in CO₂ due to binding of C_i to CmpA, excluding the dilution effect, was calculated by subtracting the data obtained by the addition of buffer from those obtained by the addition of protein solution (Fig. 2B). From the curves, the initial rates of decrease in CO₂ concentration were calculated to be 0.118 µM/s and 0.0152 µM/s in the presence and absence of CA, respectively. When 50 µM NaHCO₃ was added to the buffer in the absence of CA, the initial rate of increase in CO₂ concentration was 0.0180 µM/s (data not shown), which was similar to the initial rate of decrease in CO₂ concentration caused by removal of 45 µM of C_i from medium due to binding to CmpA. Furthermore, a hypothetical CO₂-binding protein would have been expected to cause a rapid and pronounced decline in the CO₂ concentration, in the absence of CA, followed by a slow return back to the equilibrium level. This clearly did not occur. These results also support the notion that it is HCO₃ that bound to CmpA.

Equilibrium Dialysis Experiments at Low C_i Concentrations-- Although the equilibrium dialysis using ¹⁴C-labeled HCO₃ showed specific binding of C_i to the CmpA protein, kinetic analysis of C_i binding was hardly possible by this method because of contamination by atmospheric ¹²CO₂. We therefore performed equilibrium dialysis with no added C_i, with continuous sparging of the dialysis buffer with N₂ gas to keep the concentrations of C_i in the buffer low, and used mass spectrometry for quantitation of ¹²C_i in the aqueous solutions. Different final C_i concentrations were obtained by varying the rate of sparging with N₂ and the volume of the dialysis buffer. The concentration of HCO₃ bound to CmpA was calculated by subtracting the total C_i concentration in the dialysis buffer from that in the protein solution. The free HCO₃ concentration was calculated from the total C_i concentration in the dialysis buffer and the fraction of HCO₃ in total C_i at 30 °C at the pH of assay buffer (74 and 96.5% at pH 7.0 and pH 7.5, respectively). The recombinant CmpA protein was found to bind HCO₃ with similar binding kinetics at pH 7.0 and pH 7.5 (Fig. 3). From the Scatchard plots (15) of the data, the dissociation constant for HCO₃ was calculated to be 5.8 and 5.0 µM at pH 7.0 and pH 7.5, respectively. The concentration of the bound HCO₃ under saturation was calculated to be 203 and 200 µM at pH 7.0 and pH 7.5, respectively. These values were similar to the protein concentration used, 220 µM, as calculated from the protein concentration of 10.9 mg/ml and the calculated molecular mass of 49,113 Da, suggesting that one molecule of protein carries one substrate-binding site.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Binding of HCO3 to the purified recombinant CmpA protein as a function of the substrate concentration. Aliquots of the solution of purified protein (10.9 mg/ml) were dialyzed separately against various volumes of buffer having pH of 7.0 (A) or 7.5 (B), with continuous sparging with N2 at various rates at 30 °C for 18 h. After the dialysis, concentrations of total Ci and free CO2 in the protein solutions and the dialysis buffer were measured with mass spectrometer. The concentration of the bound HCO3 [HCO3]bound) was plotted against that of free HCO3. Insets, Scatchard plots of the data.

Inhibition of PEPCase Activity with the Recombinant CmpA Protein-- Fig. 4A compares the dependence of PEPCase activity on HCO₃ concentration in assay buffer in the absence and presence of the recombinant CmpA protein. In the absence of CmpA, the maize PEPCase preparation used in this study showed a saturation kinetics with a K_m(HCO₃) value of 32 µM, which was similar to the previously reported value (20 µM) (16). In accordance with the ability of CmpA to bind HCO₃ with high affinity, the presence of CmpA was found to competitively inhibit the PEPCase reaction. The inhibitory effect was more prominent at lower concentrations of HCO₃, resulting in a sigmoidal response of the rate of PEPCase reaction on the HCO₃ concentration. At a given concentration of total HCO₃ in the CmpA solution, the concentration of free HCO₃ was obtained as the HCO₃ concentration that supported the same rate of PEPCase reaction in the absence of CmpA. The concentration of CmpA-bound HCO₃ was then calculated by subtraction of the free HCO₃ concentration from the total HCO₃ concentration. Fig. 4B shows the relationship between the concentrations of free HCO₃ and CmpA-bound HCO₃ thus obtained. From the Scatchard plot (15) of the data (Fig. 4B, inset), the dissociation constant was calculated to be 5.4 µM. The calculated concentration of the bound HCO₃ under saturation was 60.5 µM. Repeated measurements using the same protein sample yielded a K_d value of 5.35 ± 0.11 µM with the concentration of the binding site being 60.6 ± 2.2 µM (n = 3), showing the reproducibility of the method for determination of the parameters. The calculated concentration of the HCO₃ binding site was close to the protein concentration used, 61 µM, as calculated from the protein concentration of 3.0 mg/ml and the molecular mass of the protein, suggesting the presence of one substrate-binding site per one molecule of the protein. These results were essentially the same as those obtained by the equilibrium dialysis analyses using a mass spectrometer as the means of C_i quantitation.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Inhibition of PEPCase activity with the recombinant CmpA protein. A, PEPCase activity in the presence () and absence () of purified CmpA protein (3.0 mg/ml). PEPCase activity was assayed at 30 °C in a buffer containing 50 mM TES-NaOH, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 0.8 mM NADH, 6 mM phosphoenolpyruvate, and 0.5 mM NaHCO3. HCO3 concentration was calculated from the decrease in NADH concentration (see "Experimental Procedures"). PEPCase activity was plotted against the concentration of HCO3. B, binding kinetics of bicarbonate to a recombinant CmpA. At a given [HCO3]total in the CmpA solution (A), [HCO3]free was evaluated as the HCO3 concentration that provided the same rate of PEPCase reaction in the absence of CmpA. [HCO3]bound was then calculated by subtraction of [HCO3]free from [HCO3]total and plotted against [HCO3]free. Inset, Scatchard plot of the data.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial ABC importers require a substrate-binding protein that has high affinity for its specific substrate (17, 18). In this study, a recombinant CmpA protein was shown to bind HCO₃ (Fig. 2), which conforms with our previous finding that the ABC transporter encoded by the cmpABCD operon acts as an inducible, high affinity transporter for HCO₃ in Synechococcus sp. strain PCC 7942 (3). The dissociation constant of the CmpA protein, approximately 5 µM for HCO₃ (Figs. 3 and 4), is low enough to account for the apparent K_m value of the HCO₃ transporter for HCO₃, 15 µM (3). These findings indicate that the cmpA gene product functions as the substrate-binding protein of the HCO₃ transporter.

The CmpA protein was originally found as a major protein of the cytoplasmic membrane of the cells grown under CO₂-limited conditions (19). It is tightly bound to the cytoplasmic membrane despite the hydrophilicity of the predicted sequence (4). In a previous study, we showed that NrtA, a paralog of CmpA, is a substrate-binding lipoprotein, which is anchored to the cytoplasmic membrane by the lipid moieties attached covalently to the N-terminal Cys residue of the mature protein (9). Maturation of the lipoproteins requires signal peptidase II for cleavage of the signal peptide from an S-glyceride derivative of prolipoproteins with the modified Cys residue at the signal cleavage site (20). Similar to the case in NrtA, the predicted amino acid sequence around the presumed signal cleavage site of CmpA, LKGC (9), conforms to the consensus sequence recognized by signal peptidase II, (L/V/I)(A/S/T/G)(G/A)C, in which one mismatch is acceptable in the first two amino acids (21). The N terminus of the mature CmpA protein, purified from the cytoplasmic membrane of low CO₂-grown Synechococcus cells, was shown to be blocked (4). The His-tagged recombinant CmpA protein was expressed as a soluble protein in E. coli (Fig. 1), confirming the hydrophilicity of the protein without the signal peptide. These observations, together with its tight binding to the membrane, suggest that the mature form of CmpA is a lipoprotein.

The NrtA protein, which functions as the substrate-binding protein of the ABC transporter encoded by the nrtABCD genes, is 46.5% identical to CmpA and binds nitrate and nitrite with high affinity (K_d = 0.3 µM) (9). A gene (cynA)² encoding another paralog of CmpA forms a gene cluster with the cynBD genes, which encode membrane components of an ABC transporter presumably involved in uptake of cyanate (NCO) (22). The CynA protein, which is 28 and 26% identical to CmpA and NrtA, respectively, is hence supposed to be the substrate-binding protein of the putative cyanate transporter. Tam and Saier (18) previously identified eight groups of substrate-binding proteins, which are distinct in primary amino acid sequences and the nature of the substrate. The CmpA, NrtA, and CynA proteins appear to constitute the ninth distinct group of substrate-binding proteins, which are involved in binding of monoanions, together with the orthologs of NrtA (NrtA of Anabaena sp. strain PCC 7120 (23, 24), Synechocystis sp. strain PCC 6803 (25), Phormidium laminosum (26), and Plectonema boryanum (27); NasF of Klebsiella pneumoneae (28)) and the putative orthologs of CmpA (Slr0040 of Synechocystis sp. strain PCC 6803 (29), the protein encoded by an open reading frame (bases 87,787-86,411 on the c374 segment of genomic DNA sequence; Cyanobase) of Anabaena sp. strain PCC 7120).

It should be also noted that there is another class of CmpA/NrtA homologs in cyanobacteria. These are the C-terminal domain of CmpC and that of NrtC. CmpC and NrtC are each one of the two ATP-binding subunits of the bicarbonate- and nitrate/nitrite transporters, respectively, and are composed of two distinct domains. Whereas their N-terminal domains are strongly similar to the ATP-binding subunits of other ABC transporters and to each other, their C-terminal domains are 30% identical to CmpA, NrtA, and to each other. The C-terminal domain of NrtC is required not for nitrate/nitrite transport but for ammonium-promoted inhibition of the transport (30), indicating that it is a regulatory domain of the transporter. The similarity of the regulatory domain of NrtC to CmpA/NrtA and the capacity of CmpA/NrtA for substrate binding suggest that the regulation of nitrate/nitrite transport might involve binding of a certain compound, probably an anion, to the regulatory domain. Although it is unknown what kind of regulation the HCO₃ transporter is subject to, it is inferred by analogy that the C-terminal domain of CmpC has a regulatory role in HCO₃ transport. Biochemical and molecular biological studies on the NrtC and CmpC proteins are being performed to elucidate the structure-function relationships of the C-terminal domains of these proteins.

	FOOTNOTES

^* This work was supported by Grant-in-aid for Scientific Research C 09640768 and Grant-in-aid for Scientific Research in Priority Areas A 09274103 (to T. O.) from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by core funding from the Research School of Biological Sciences, Institute of Advanced Studies, Australian National University.

To whom correspondence should be addressed: Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Tel.: 81-52-789-4106; Fax: 81-52-789-4107; E-mail: omata@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, April 21, 2000, DOI 10.1074/jbc.M003034200

² F. Jalali and G. S. Espie, GenBank^TM accession no. AF001333.

	ABBREVIATIONS

The abbreviations used are: C_i, inorganic carbon; PEPCase, phosphoenolpyruvate carboxylase; CA, carbonic anhydrase; IPTG, isopropyl-1-thio--D-galactopyranoside; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. D. Price, M. R. Badger, F. J. Woodger, and B. M. Long Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation and prospects for engineering into plants J. Exp. Bot., May 1, 2008; 59(7): 1441 - 1461. [Abstract] [Full Text] [PDF]

				G. S. Espie, F. Jalali, T. Tong, N. J. Zacal, and A. K.-C. So Involvement of the cynABDS Operon and the CO2-Concentrating Mechanism in the Light-Dependent Transport and Metabolism of Cyanate by Cyanobacteria J. Bacteriol., February 1, 2007; 189(3): 1013 - 1024. [Abstract] [Full Text] [PDF]

				N. M. Koropatkin, D. W. Koppenaal, H. B. Pakrasi, and T. J. Smith The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA J. Biol. Chem., January 26, 2007; 282(4): 2606 - 2614. [Abstract] [Full Text] [PDF]

				F. J. Woodger, M. R. Badger, and G. D. Price Inorganic Carbon Limitation Induces Transcripts Encoding Components of the CO2-Concentrating Mechanism in Synechococcus sp. PCC7942 through a Redox-Independent Pathway Plant Physiology, December 1, 2003; 133(4): 2069 - 2080. [Abstract] [Full Text]

				P. Chain, J. Lamerdin, F. Larimer, W. Regala, V. Lao, M. Land, L. Hauser, A. Hooper, M. Klotz, J. Norton, et al. Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea J. Bacteriol., May 1, 2003; 185(9): 2759 - 2773. [Abstract] [Full Text] [PDF]

				M. R. Badger and G. D. Price CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution J. Exp. Bot., February 1, 2003; 54(383): 609 - 622. [Abstract] [Full Text] [PDF]

				F. Huang, I. Parmryd, F. Nilsson, A. L. Persson, H. B. Pakrasi, B. Andersson, and B. Norling Proteomics of Synechocystis sp. Strain PCC 6803: Identification of Plasma Membrane Proteins Mol. Cell. Proteomics, December 1, 2002; 1(12): 956 - 966. [Abstract] [Full Text] [PDF]

				T. Omata, S. Gohta, Y. Takahashi, Y. Harano, and S.-i. Maeda Involvement of a CbbR Homolog in Low CO2-Induced Activation of the Bicarbonate Transporter Operon in Cyanobacteria J. Bacteriol., March 15, 2001; 183(6): 1891 - 1898. [Abstract] [Full Text]

				C. V. Hoang and K. D. Chapman Biochemical and Molecular Inhibition of Plastidial Carbonic Anhydrase Reduces the Incorporation of Acetate into Lipids in Cotton Embryos and Tobacco Cell Suspensions and Leaves Plant Physiology, April 1, 2002; 128(4): 1417 - 1427. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20551    most recent M003034200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maeda, S.-i. Articles by Omata, T. Search for Related Content PubMed PubMed Citation Articles by Maeda, S.-i. Articles by Omata, T. Social Bookmarking                      What's this?