Association of SbtA and cognate SbtB proteins in response to adenylnucleotides.

The SbtA7001-SbtB7001 and SbtA7942-SbtB7942 pairs were expressed in E. coli (A, C) and Synechococcus ΔCS (denoted by *; B, D). The HAHis-tagged SbtB proteins were immobilized to examine interaction with SbtA proteins and adenylnucleotides (2 mM ATP, ADP, AMP, or cAMP) during in vitro IMAC binding assays. The HAHis-SbtB protein and corresponding amounts of SbtA retained in SbtA:SbtB complexes were detected by immunoblot analyses. Representative results are shown of n=3-8 experiments per adenylnucleotide treatment.

The association of SbtA and cognate SbtB proteins is not directly linked to key metabolites of the CBB cycle (RUBP, 3PGA) and the TCA cycle (2OG).

In vitro IMAC binding assays were performed with the SbtA7001-SbtB7001 (A) and SbtA7942-SbtB7942 (B) pairs expressed in E. coli in the presence of 2 mM RUBP, 3PGA or 2OG, and 2 mM 2OG combined with 2 mM ADP, or ATP (2OG ADP and 2OG ATP, respectively). The representative immunoblot images show HAHis-tagged SbtB proteins and the SbtA proteins retained in SbtA:SbtB complexes; n=2.

Effects of ATP:ADP and ATP:AMP ratios on the association of SbtA and SbtB proteins.

The SbtA7001-SbtB7001 (A, C, E) and SbtA7942-SbtB7942 (B, D, F) pairs were expressed in E. coli (A, C, E, F) and in Synechococcus ΔCS (denoted by *; B, D). Variation in energy charge was simulated during in vitro IMAC binding assays by different molar ratios of ATP:ADP (A-D) and ATP:AMP (E, F), with a total adenylnucleotide concentration of 2 mM. Representative immunoblot images of HAHis-tagged SbtB and SbtA proteins retained in SbtA:SbtB complexes of n=3-4 per adenylnucleotide treatment. The 17 kD-protein above SbtB7942 is a fragment occasionally detected with the anti-SbtA antibody (C, F).

Effects of the cAMP:ADP and cAMP:AMP ratios on the association of SbtA and SbtB proteins.

In vitro IMAC binding assays were performed across a range of cAMP:ADP (A, B) and cAMP:AMP (C, D) ratios, with a total adenylnucleotide concentration of 2 mM. The SbtA7001-SbtB7001 (A, C) and SbtA7942-SbtB7942 (B, D) pairs were expressed in E. coli. The immunoblot images show HAHis-tagged SbtB and SbtA proteins retained in SbtA:SbtB complexes and are representative of three independent experiments per adenylnucleotide treatment.

Quantitative estimates for the association of SbtA and SbtB proteins in response to different adenylnucleotide ratios (AR).

The ratios of ATP:ADP or AMP (A) and cAMP:ADP or AMP (B) that supported 50% (AR50) and 10% (AR10) of SbtA bound to SbtB were estimated from sigmoidal logistic curve fits (Fig. S4). AR values are means (± SE) of 3-4 independent experiments. AR values were not significantly different across each AR category at a P=0.05 threshold (AR50 P=0.232, AR10 P=0.275).

Light-dependent bicarbonate uptake by SbtA is modulated by SbtB in Synechococcus ΔCS.

The SbtA7942 protein was expressed alone (SbtA) or with SbtB7942 (SbtAB) in Synechococcus ΔCS (ΔCS) at low Ci in the light. CO2 uptake was inhibited by EZ to restrict Ci acquisition to bicarbonate uptake by SbtA. (A) HCO3- uptake-dependent gross photosynthetic O2 evolution rates in response to stepwise increased light intensity after 1 h dark-adaptation. Respiratory O2 consumption was the same for SbtA and SbtAB (27 ± 4 and 27 ± 3, respectively). Values are means ± SE; n=4-5. (B) Induction of HCO3- transport activity of SbtA in dark-adapted cells immediately after dark-to-low light (30 μmol m-2 s-1) transitions at pH 8 and pH 9 during 30-60 s pulses of [14C]HCO3- uptake, using silicon oil centrifugation-filtration. HCO3- uptake rates are shown relative to cells expressing SbtA alone; n=4-5. Relative HCO3- uptake rates were calculated by subtraction of the unspecific incorporation of 14C-label, mainly due to extracellular [14C] HCO3- in the water shell surrounding each cell and a very small amount of 14CO2 diffusion into the cells. This was derived from 14C-label incorporation into Ci uptake inhibited ΔCS (38% ± 6 (pH 8) and 42% ± 8 (pH 9). (C) HCO3- uptake rates normalized to Chla content at various time points after a light (400 μmol m-2 s-1) to dark shift. Values are means ± SE; n=3-5. Statistically significant differences (P < 0.05) are denoted by an asterisk (A) or different letters (B).

Structural comparison of the overall fold of SbtA and SbtB protein models.

(A) Superposition of homology models of SbtA7001 (light orange) and SbtA7942 (light blue). The core domains and arrangement of the transmembrane helices are well matched but the 5/6 loop connecting transmembrane helix 5 and 6 is divergent, protruding further into the cytoplasm on SbtA7942. (B) Superposition of the three-dimensional models based on crystal structures of SbtB7001 with Ca2+ and ATP ligands (red-orange, SbtB7001-CaATP; PDB: 6MM2) and SbtB7001 with ADP bound (orange, SbtB7001-ADP; PDB: 6MMC), demonstrating that ligand-induced structural differences affect primarily the T-loop. The T-loop assumes a closed conformation in SbtB7001-CaATP and an open, disordered state in SbtB7001-ADP which was not resolved in crystals as indicated by the dashed line. (C) Superposition the of the homology model of the apo-SbtB7942 monomer (blue) on SbtB7001-CaATP; both SbtB proteins are predicted to be nearly identical in three-dimensional structures. (D) Superposition of the SbtA7001 and SbtB7001-CaATP onto a SbtA6803 monomer (PDB: 7EGK, chain C) associated with a SbtB680-AMP monomer (chain D) which were extracted from the trimeric SbtAB6803 cryo-EM structure (PDB: 7EGK). Since the three-dimensional architecture of SbtA7001 and SbtA6803 are highly similar (Fig. S12), only SbtA7001 is shown for clarity. The core structure of SbtB7001-CaATP and SbtB6803-AMP is nearly identical, but the T-loop conformations diverge in a ligand-specific manner consistent with the observed SbtA:SbtB interaction in this study (enlarged). Binding of AMP to SbtB6803 (green) promotes an open T-loop conformation which extends into SbtA consistent with SbtA:SbtB association, whereas Ca2+ATP bound to SbtB7001 (red-orange) promotes folding of the T-loop away from SbtA.

Proposed model for regulation of SbtA bicarbonate transport activity involving SbtB by environmental stimuli and the adenylate energy charge (AEC).

In the light, expression of SbtA and SbtB in Synechococcus elongatus is induced by low Ci availability. The plasma membrane (pm)-integral SbtA trimer(SbtA3) actively transports bicarbonate from the external environment (ext) into the cytoplasm (cyto) presumably dissociated from SbtB. SbtA activity depends on Na+-symport and requires a cell-inward-directed Na+ gradient with a probable HCO3-:Na+ symport stoichiometry of 1:1 through each monomer. Light-stimulated photosynthetic electron transport sustains high levels of intracellular ATP over ADP and AMP, i.e. high AEC, which facilitates binding of Ca2+ATP to SbtB trimers (SbtB3), stabilizing the SbtB T-loops, and SbtB remains dissociated from SbtA. In prolonged darkness, cells operate at a lower AEC. Relatively higher amounts of ADP and AMP replace ATP as ligands in SbtB trimers, which favours open T-loops, and inactive SbtA:SbtB complexes are formed. In such scenario, SbtA reactivation after a transition from dark to light would depend on the rate of increase in AEC and concomitant AEC-dependent dissociation of SbtB.

Primers for generation of the bicarbonate transporter-deficient Synechococcus ΔCS mutant.

E. coli and cyanobacterial cell lines and plasmids used for protein expression and physiological analyses of the SbtA and SbtB proteins from Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942.