Adenylnucleotide-mediated binding of the PII-like protein SbtB contributes to controlling activity of the cyanobacterial bicarbonate transporter SbtA
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eLife assessment
This study is of fundamental importance, addressing the regulation of the carbon concentrating mechanism in cyanobacteria. It is a well-controlled investigation of low affinity regulatory binding of small molecules, processes that are typically difficult to examine. The work provides compelling evidence that the adenylate pool, rather than any single metabolite, regulates a key bicarbonate transporter (SbtA) to provide efficient bicarbonate supply while preventing futile cycling that can result from escape of unfixed CO2.
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Abstract
Cyanobacteria have evolved a remarkably powerful CO2 concentrating mechanism (CCM), enabling high photosynthetic rates in environments with limited inorganic carbon (Ci). Therefore, this CCM is a promising system for integration into higher plant chloroplasts to boost photosynthetic efficiency and yield. The CCM depends on active Ci uptake, facilitated by bicarbonate transporters and CO2 pumps, to elevate CO2 concentration around the active sites of the primary CO2 fixing enzyme, Rubisco, which is encapsulated in cytoplasmic micro-compartments (carboxysomes). The essential CCM proteins have been identified, but the molecular signals and regulators that coordinate function in response to light, Ci availability and other environmental cues are largely unknown. Here, we provide evidence, based on a novel in vitro binding system, for a role of the PII-like SbtB protein in regulating Ci uptake by the bicarbonate transporter, SbtA, in response to the cellular adenylate energy charge (AEC) through dynamic protein-protein interaction. Binding of the SbtA and SbtB proteins from two phylogenetically distant species, Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942, was inhibited by high ATP, and promoted by low [ATP]:[ADP or AMP] ratios in vitro, consistent with a sensory response to the AEC mediated through adenylnucleotide ligand-specific conformation changes in SbtB. In vivo, cell cultures of S. elongatus showed up to 70% SbtB-dependent down-regulation of SbtA bicarbonate uptake activity specifically in the light activation phase during transitions from dark to low light when low cellular AEC is expected to limit metabolic activity. This suggests SbtB may function as a curfew protein during prolonged low cellular AEC and photosynthetically unfavourable conditions to prevent energetically futile and physiologically disadvantageous activation of SbtA.
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eLife assessment
This study is of fundamental importance, addressing the regulation of the carbon concentrating mechanism in cyanobacteria. It is a well-controlled investigation of low affinity regulatory binding of small molecules, processes that are typically difficult to examine. The work provides compelling evidence that the adenylate pool, rather than any single metabolite, regulates a key bicarbonate transporter (SbtA) to provide efficient bicarbonate supply while preventing futile cycling that can result from escape of unfixed CO2.
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Reviewer #1 (Public Review):
Using an immobilised metal affinity chromatography (IMAC)-based assay coupled with Western blot immunodetection analysis, SbtB, the regulatory protein for SbtA activity, is shown in itself to be regulated by the local adenylate energy charge (AEC), with inhibitory binding of SbtB to SbtA disfavoured at high ATP:ADP ratios. Such conditions are expected to be encountered during steady-state photosynthesis with the associated cellular demand for Ci and SbtA activity.
By homology with ATP-binding PII proteins, ATP is proposed to interact with a loop region of SbtB, changing its conformation on binding and inhibiting the formation of the (inactive) SbtA:SbtB complex. On the basis of this, the authors propose that SbtB acts an AEC-sensing 'curfew' protein for SbtA activity, tuning bicarbonate import by this …
Reviewer #1 (Public Review):
Using an immobilised metal affinity chromatography (IMAC)-based assay coupled with Western blot immunodetection analysis, SbtB, the regulatory protein for SbtA activity, is shown in itself to be regulated by the local adenylate energy charge (AEC), with inhibitory binding of SbtB to SbtA disfavoured at high ATP:ADP ratios. Such conditions are expected to be encountered during steady-state photosynthesis with the associated cellular demand for Ci and SbtA activity.
By homology with ATP-binding PII proteins, ATP is proposed to interact with a loop region of SbtB, changing its conformation on binding and inhibiting the formation of the (inactive) SbtA:SbtB complex. On the basis of this, the authors propose that SbtB acts an AEC-sensing 'curfew' protein for SbtA activity, tuning bicarbonate import by this protein for situations when carbon fixation would be physiologically (and energetically) advantageous. As SbtA is a HCO3-/Na+ symporter, Na+ homeostasis would also be controlled by regulation of this transporter.
The IMAC assay used to monitor SbtA:SbtB complex stability as a function of AEC seems robust, is relatively straightforward and may be of interest to other researchers investigating adenylate-sensing protein reaction partners (with the usual caveats on extrapolating in vitro results to living systems, as noted by the authors).
In this study, SbtA regulation was also investigated in vivo in a Synechococcus HCO3- transporter knockout mutant via measurement of labelled HCO3- uptake and overall photosynthetic performance (MIMS-monitored O2 evolution as a function of PAR). Here, SbtB was inferred to regulate SbtA activity during the induction of photosynthesis (i.e. at low ATP:ADP) and not when photosynthesis was fully activated and in a steady-state condition. SbtA inactivation on a light-dark transition was also demonstrated in vivo irrespective of the presence SbtB, indicative of additional regulatory pathways affecting the activity of this transporter. These conclusions seem to be well-supported by the presented data.
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Reviewer #2 (Public Review):
Inorganic carbon (Ci) uptake by autotrophic organisms is often the rate-limiting process in overall photosynthetic productivity. Aquatic autotrophs including the cyanobacteria have evolved elaborate and metabolically expensive, yet very efficient CO2 concentrating mechanisms (CCMs) to over-come this limitation. The work examines the regulation of SbtA, which is a high affinity sodium dependent symporter. Current evidence suggests that this SbtA is highly regulated both at the transcriptional and post-transcriptional levels. For example, the sbtA gene is transcriptionally upregulated under conditions of inorganic carbon limitation and the transport activity of the expressed SbtA protein is apparently regulated allosterically by multiple factors, including those exerted by the binding of the small trimeric …
Reviewer #2 (Public Review):
Inorganic carbon (Ci) uptake by autotrophic organisms is often the rate-limiting process in overall photosynthetic productivity. Aquatic autotrophs including the cyanobacteria have evolved elaborate and metabolically expensive, yet very efficient CO2 concentrating mechanisms (CCMs) to over-come this limitation. The work examines the regulation of SbtA, which is a high affinity sodium dependent symporter. Current evidence suggests that this SbtA is highly regulated both at the transcriptional and post-transcriptional levels. For example, the sbtA gene is transcriptionally upregulated under conditions of inorganic carbon limitation and the transport activity of the expressed SbtA protein is apparently regulated allosterically by multiple factors, including those exerted by the binding of the small trimeric protein, SbtB. SbtB is a PII-type regulator that conditionally binds to the cytoplasmic face of the trimeric SbtA to form a hetero-complex apparently inactivating SbtA to which it is bound. The factors affecting this interaction remains to be clarified, but it is already clear that there is considerable complexity that needs to be unraveled since as with other PII proteins, multiple effector molecules act as ligands.
Using a novel protein-protein interaction assay combined with physiological analysis of various mutants, the authors present new information on the regulation of SbtA from Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942. Because of their novelty, additional validation may be important to establish their validity, yet they do appear to be robust overall..The work builds on earlier studies indicating negative regulation of SbtA and helps clarify other work, including detailed analysis of the orthologous, albeit somewhat more complex protein from Synechocystis PCC6803. The key significance of the present findings is that the energy charge of the adenylate system, a ubiquitous metabolic control mechanism in the biological world, is the prime and perhaps overriding regulatory parameter governing of SbtA activity. Based on this a model for the diurnal control transporter activity was proposed based on energy charge.
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Reviewer #3 (Public Review):
The regulation of transporters in many physiological systems is poorly known. Here, Forster and colleagues describe how activity of an inorganic carbon transporter, SbtA, in the bacterial carbon concentrating mechanism is regulated by the PII protein SbtB. Although there is now significant structural knowledge of the system and many potential SbtB-regulating small molecule effectors are known, Forster and colleagues clarify, how the adenylate charge in the cell, rather than any single metabolite, is the important regulatory effector. This is critical for the endogenous function, as the cyanobacterial host undergoes dramatic changes in adenylate charge over the course of a diurnal cycle and this result explains how the channel is regulated to efficiently function in CO2 assimilation. The manuscript is …
Reviewer #3 (Public Review):
The regulation of transporters in many physiological systems is poorly known. Here, Forster and colleagues describe how activity of an inorganic carbon transporter, SbtA, in the bacterial carbon concentrating mechanism is regulated by the PII protein SbtB. Although there is now significant structural knowledge of the system and many potential SbtB-regulating small molecule effectors are known, Forster and colleagues clarify, how the adenylate charge in the cell, rather than any single metabolite, is the important regulatory effector. This is critical for the endogenous function, as the cyanobacterial host undergoes dramatic changes in adenylate charge over the course of a diurnal cycle and this result explains how the channel is regulated to efficiently function in CO2 assimilation. The manuscript is generally clear and the data generally supportive of the conclusions as written. However, there are several instances where additional clarification and/or experiments are needed to confirm the major findings of the paper.
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