Scaffolding protein CcmM directs multiprotein phase separation in β-carboxysome biogenesis

Carboxysomes in cyanobacteria enclose the enzymes Rubisco and carbonic anhydrase to optimize photosynthetic carbon fixation. Understanding carboxysome assembly has implications in agricultural biotechnology. Here we analyzed the role of the scaffolding protein CcmM of the β-cyanobacterium Synechococcus elongatus PCC 7942 in sequestrating the hexadecameric Rubisco and the tetrameric carbonic anhydrase, CcaA. We find that the trimeric CcmM, consisting of γCAL oligomerization domains and linked small subunit-like (SSUL) modules, plays a central role in mediation of pre-carboxysome condensate formation through multivalent, cooperative interactions. The γCAL domains interact with the C-terminal tails of the CcaA subunits and additionally mediate a head-to-head association of CcmM trimers. Interestingly, SSUL modules, besides their known function in recruiting Rubisco, also participate in intermolecular interactions with the γCAL domains, providing further valency for network formation. Our findings reveal the mechanism by which CcmM functions as a central organizer of the pre-carboxysome multiprotein matrix, concentrating the core components Rubisco and CcaA before β-carboxysome shell formation.

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-In the abstract, please point out the key feature of gCAL(CcmM), which enables network formation -Please provide schematic representation of the different domains -Please introduce condensates and multivalent interactions in intro (PMID 22398450, 29602697) -What is the role of condensate formation in Rubisco?
-Results: provide more details on the material state of condensates (gel, amyloid -like etc.) based on experimental data -Results: it would be useful to support the analysis on C2 motif by bioinformatics predictions to see the interplay between hydrophobic interactions and protein dynamics (PMID 33318217) in particular, that a disordered motif drives formation of a non-mobile association -Here the critical concentration is ~micromol, and the interaction affinity is also in the same order of magnitude. In most multivalent systems, the individual motifs are ~mM (see PMID 27203110). Can you comment on this? -How the multivalent interactions seen here relate to those in ref 16? -Results: can you provide the KD for the full protein to see how much the flanking regions contribute to interactions? -Results: in the structure part the direct link between the observed interactions and multivalency is not clear -What is the role of disordered flanking regions and protein dynamics? Cryo-EM indicates protruding densities, which might be critical for the network assembly .
-The role of dynamics is not clear in the discussion it is mentioned as more dynamical state, in the results as a not dynamic association. Is it possible that salt concentration also affects the dynamics of protein chains?
References -cite head-to-tail polymerisation as a mechanism for phase separation (PMID 32822572) or for less dynamic assemblies (PMID 27203110) Reviewer #2: Remarks to the Author: Summary of the key results: The proteinaceous microcompartments known as carboxysomes found in cyanobacteria have been a great interest to the photosynthesis research community because these bacterial organelles are highly effective in concentrating CO2 around Rubisco. In β-carboxysomes, the main scaffolding protein, CcmM, is known to function as two isoforms: M58, which is the full-length version, and M35, which is the shorter version with only the C-terminal part of M58. Previous studies showed that both isoforms are essential components of β-carboxysomes. M35 is made up of three repeats of Rubisco small subunit-like (SSUL) modules, and previous work (Wang et al, Nature, 566, 131-135, 2019) detailed how it forms condensate with Rubisco through structural and biochemical methods. The N-terminal part of M58 contains a γ carbonic anhydrase-like domain (γ-CAL) and has been known to interact with at least two other proteins of β-carboxysomes, CcmN and βCA. However, the detailed mechanism of the interecations between M58 and other proteins are not known. In this manuscript, Zang et al describe their findings from biochemical and structural studies of M58, βCA, Rubisco and M35. They found that M58 is able to form three different types of condensates: with βCA, Rubisco or by itself. The biochemical and structural experiments are well designed and clearly presented in the manuscript. Not only did they address the critical interaction between M58 and βCA at the atomic levels, they also revealed two unexpected interactions: one between the SSUL modules and γ-CAL domain of M58 and the head-tohead interaction between γ-CAL domains from two M58 trimers. All these interactions lead to intricate network of proteins influenced by redox conditions. Hence, this manuscript is an important step towards understanding the detailed interactions among proteins inside β-carboxysomes and should be of great interest to the researchers studying β-carboxysomes as well as implementing them into plants for bioengineering purposes. Despite the intricate nature of the materials, the manuscript is well organized, and the findings are clearly described.
Minor suggestions for the authors: 1. In line 202, when homo-demixing of M58 in Fig. 4c,d was described, it would be helpful to point out the difference betweeen the experiments in  , 27, 2637-2644, 2015) previously showed that a single chimeric protein was able to structurally and functionally replace four proteins: M58, M35, βCA and CcmN. The chimeric protein was derived by replacing γCAL in M58 with a shorter version of βCA. It was not understood how the chimeric protein was able to replace both M58 and M35. Would the new findings in the current manuscript somehow provide some 'speculative' explanation regarding the unusual behavior of that chimeric protein? For example, the current manuscript suggests there is differential redox-regulation of the interactions between Rubisco and M58 versus M35. More specifically, the interaction between SSUL modules and γCAL in M58, which appeared enhanced under oxidizing conditions, is no longer present with the chimeric protein. Could the redox regulation of the chimeric protein be more similar to M35, while at the same time, it is more structurally and physically similar to M58? Can the authors provide some insights into the chimeric protein in Gonzalez-Esquer et al paper in the discussion section?
Reviewer #3: Remarks to the Author: Carboxysome is a special microcompartment formed in cyanobacteria and algae that enclose the enzymes Rubisco and carbonic anhydrase to optimize photosynthetic carbon fixation. Understanding the biogenesis of carboxysome assembly and implementing an optimized carboxysome-like CO2 concentrating mechanism (CCM) into plants is a desirable strategy to increase the crop yield. In this study, the authors revealed a central role of the scaffolding protein CcmM M58 via initiating phase separation in the biogenesis of -carboxysomes in cyanobacteria. Specially, the N-terminal CAL domain of M58 mediate interactions with CcaA (support the CA activity), intermolecular interactions with the SSUL domain, and also head-to-head association of CcmM trimers. Overall, this study provides new sights into the biogenesis of -carboxysome. I have a few concerns on the manuscript. 1. In figure 2f and 3, it seems that W257 in the C2 peptide play crucial roles in mediating the interactions with the CAL domain of M58. Further biochemical assays should be carried out to validate the structural model as well as the key involved residues.
2. CcaA exhibits as a tetramer in solution ( Fig S1). It seems the preceding C1 peptide mediates the oligmerization. In the complex structure of CAL-C2, each protomer of CAL binds a C2 peptide. The conclusion "each protomer of the γCAL trimer interacts with one protomer of a CcaA tetramer" should be biochemically validated using full length proteins. 3. In Supplemental Fig 6, for the 3.6 Å resolution map, the color gradient of the local resolution maps of side and end views from blue to red indicates local resolution from 2.0 to 6.0 Å. I suggest the authors show the values in a shorter range (3.0 to 6.0 Å?). 4. In Supplemental Fig 8, the authors showed the structural model of M58-RbcL8 to further validate the interaction between M58 and Rubisco complex. As the resolution of M58-RbcL8 is only 8 Å, information provided from the model is very limited and not reliable. I think this part is not necessary in the current manuscript. 5. In supplementary Table 3, the authors should provide more parameters to show the model validations. 6. In figures 1, 4, 7 and supplementary figs 2, 4, 10, the authors show the condensates of different proteins. In some figures, the authors exhibited the FRAP assays. It will be better to additionally show the results of droplet fusion. 7. The authors demonstrated that Rubisco, M58, M35 and CcaA could co-assembly in vitro. How about in vivo? It will be better to confirm the co-assembly in vivo. 8. In figure 6, the authors demonstrated that trimeric M58 binds rubisco with high affinity in a turbidity assay. Maybe some additional assays such as ITC will be better. 9. CcmM directs multiprotein phase separation in β-carboxysome biogenesis. How about the effect of this phase separation on carbon fixation? Whether the ability of β-carboxysome carbon fixation depends on this phase separation?

Author Rebuttal to Initial comments
Reviewer #1 (phase separation) Remarks to the Author: The paper provides insights into the molecular interactions underlying of the complex network of Rubisco, leading to condensate formation. While some of the details are highly elaborated, several key issues remained to be elusive regarding the material state of the assembly, the connection between dynamics and condensate formation, the role of motif-flanking regions in mediating multivalent interactions. The paper would also benefit from relating the findings to other relevant papers in the field, regarding Rubisco multivalency, condensate formation via head-to-tail mechanisms, thermodynamics and material states with multivalent higher-order systems, as listed below.
Thank you for focusing our attention on these points. We agree that a more extensive discussion of our findings regarding Rubisco in light of the known biophysical principles of condensate formation will make our study more attractive for a broad readership. We have amended the Introduction and Discussion sections accordingly.
-In the abstract, please point out the key feature of gCAL(CcmM), which enables network formation We have rewritten the abstract to emphasize the role of the γCAL domains of M58 in sequestering Rubisco and CcaA via multivalent interactions.
-Please provide schematic representation of the different domains. "When you read this paper being not from the RUbisco field, it is difficult to capture the jargon of names. I had difficulties synchronizing the names with the text, in particular in this format. I think some names appear before their scheme.
Following the reviewer's suggestion, we have added the structure of Rubisco and schematics of M58. M35 and CcaA in Fig. 1.
-Please introduce condensates and multivalent interactions in intro (PMID 22398450, 29602697) We have done as suggested, and have cited these and other papers.
-What is the role of condensate formation in Rubisco?
Condensate formation of Rubisco, CcaA and other proteins, involved in carbon fixation, is the first step in the biogenesis of carboxysomes. This is mediated by the scaffolding protein CcmM (M58 and M35 in Synechococcus elongatus PCC 7942). Once the carboxysome proteinaneous shell has formed, the material property of the condensate is no longer critical to maintain the compartment. Thus, metabolic compartments, including carboxysomes, differ from membraneless condensates, such as stress granules or the nucleolus which do not have a shell and are dissociable. In fact, some of the multivalent interactions in the pre-carboxysome condensate are weakened (Wang H et al., Nature 2019) after shell formation, as the interior of the carboxysome is oxidizing (Price GD et al., Plant Physiol. 1992;Peña KL et al., PNAS 2010;Chen AH et al., PLoS One 2013). These aspects are now discussed in more detail.
-Results: provide more details on the material state of condensates (gel, amyloid -like etc.) based on experimental data By microscopic observation the condensate is viscous, but it is not a "hydro-gel" or amyloid-like aggregate. Consistent with a state of low liquidity, the droplets of the 4-protein condensate (Rubisco, M58, M35 and CcaA) undergo slow fusion. This is now shown in new Fig. 7e and Supplementary Movie 5. Moreover, Rubisco in the condensate remains fully enzymatically active (new Fig. 7g), excluding an amyloid/fibrillar state.
-Results: it would be useful to support the analysis on C2 motif by bioinformatics predictions to see the interplay between hydrophobic interactions and protein dynamics (PMID 33318217) in particular, that a disordered motif drives formation of a non-mobile association The FuzDrop method developed by the group of Vendruscolo (Hardenberg M et al., PNAS 2020) is based on the assumption that a droplet state is stabilized by non-specific or low-specific side-chain interactions. This does not apply to the C2-γCAL interaction, which is based on specific interactions with a welldefined interface, as seen in the crystal structure. The C2 motif binds as a two-turn helix. We now show that mutating Trp257 to Ala and Arg265 to Asp in the C2 sequence of CcaA reduced condensate formation with M58 by ~50%, or completely abolished condensate formation, respectively, as analyzed in the turbidity assay (see new Fig. 3d). Furthermore, both mutations in EGFPC17 abolished complex formation with γCAL by gel-shift assay (see new Supplementary Fig. 3i). These results underscore the specific contribution of hydrophobic and charge interactions to binding as indicated by the crystal structure.
-Here the critical concentration is ~micromol, and the interaction affinity is also in the same order of magnitude. In most multivalent systems, the individual motifs are ~mM (see PMID 27203110). Can you comment on this?
Thank you for pointing this out. As explained above, the interactions involved in Rubisco/CcaA condensation by CcmM rely on specific interactions and occur with relatively high affinity compared to the more fluctuating interactions underlying the formation of membraneless condensates. This makes sense in order to ensure that Rubisco and CcaA are concentrated with high efficiency, limiting escape and ensuring efficient encapsulation upon shell formation. This is now emphasized in the revised manuscript.
-How the multivalent interactions seen here relate to those in ref 16?
There are interesting differences and similarities between the Rubisco-CsoS2 and the Rubisco-CcmM interactions. The main difference is that in case of CsoS2 the interaction is mediated by 4 disordered repeat motifs, while the SSUL modules of M58/M35 interact as folded domains. Multivalency is critical in both interactions and is highly efficient in capturing Rubisco, involving similar binding regions on Rubisco. Given the space limitations, a detailed discussion of the paper by Oltrogge et al. would need to be deferred to a future review article. However, we have inserted a statement that α-carboxysomes use intrinsically disordered, linear motifs as phase separation scaffold.
-Results: can you provide the KD for the full protein to see how much the flanking regions contribute to interactions?
The apparent KD values determined by turbidity assay for the interactions of the full proteins, previously in the text, are now shown in Fig. 1f and Fig. 6a. Note that ITC measurements are not possible for condensate forming reactions. The flanking regions flexibly linking the SSUL modules show no conserved motif or sequence. These sequences are not resolved in the cryo-EM structure of the M35-Rubisco condensate (Wang H et al., Nature 2019). It seems unlikely that they contribute directly to the interaction, although this cannot be completely ruled out. A detailed analysis of their role is beyond the scope of the present study.
-Results: in the structure part the direct link between the observed interactions and multivalency is not clear.
The structural model of the multivalent interaction between the γCAL trimer and three C2 peptide sequences of CcaA, based on the crystal structure and on the binding stoichiometry determined by ITC in solution, is now shown as a main figure . Since each γCAL trimer binds one CcaA subunit of a CcaA tetramer, this indicates, in principle, that one γCAL trimer has the ability to interact with three CcaA tetramers, resulting in a multivalent network. Whether three tetramers bind simultaneously would depend on steric effects and the dynamics of the interaction. We have explained this more clearly in the revised manuscript.
-What is the role of disordered flanking regions and protein dynamics? Cryo-EM indicates protruding densities, which might be critical for the network assembly .
Please see point above. The protruding densities observed in the cryo-EM map corresponds to SSUL modules. This interaction is indeed critical for M58 homo-demixing as shown by the mutational analysis in Fig. 4f.
-The role of dynamics is not clear in the discussion it is mentioned as more dynamical state, in the results as a not dynamic association. Is it possible that salt concentration also affects the dynamics of protein chains?
Our study focuses on the mechanism of pre-carboxysome formation which occurs in the reducing environment of the cytosol. In the Discussion, we suggested a potential redox regulation, where condensate dynamicity might increase after shell formation within the oxidizing interior of the carboxysome (see Introduction and Fig. 1b). Yes, indeed, salt concentration does affect condensate formation (see Fig. 1d,e, Supplementary Fig. 4f, Supplementary Fig. 7a-d). Thus, differences in salt concentration between the cytosol and the carboxysome interior could affect condensate dynamicity. There is, however, no information available whether such a difference exists.

References
-cite head-to-tail polymerisation as a mechanism for phase separation (PMID 32822572) or for less dynamic assemblies (PMID 27203110) Thank you for bringing these references to our attention, they are now included in the revised manuscript.
Reviewer #2 (photosynthesis, carboxysomes) Remarks to the Author: Summary of the key results: The proteinaceous microcompartments known as carboxysomes found in cyanobacteria have been a great interest to the photosynthesis research community because these bacterial organelles are highly effective in concentrating CO2 around Rubisco. In β-carboxysomes, the main scaffolding protein, CcmM, is known to function as two isoforms: M58, which is the full-length version, and M35, which is the shorter version with only the C-terminal part of M58. Previous studies showed that both isoforms are essential components of β-carboxysomes. M35 is made up of three repeats of Rubisco small subunit-like (SSUL) modules, and previous work (Wang et al, Nature, 566, 131-135, 2019) detailed how it forms condensate with Rubisco through structural and biochemical methods. The N-terminal part of M58 contains a γ carbonic anhydrase-like domain (γ-CAL) and has been known to interact with at least two other proteins of β-carboxysomes, CcmN and βCA. However, the detailed mechanism of the interecations between M58 and other proteins are not known. In this manuscript, Zang et al describe their findings from biochemical and structural studies of M58, βCA, Rubisco and M35. They found that M58 is able to form three different types of condensates: with βCA, Rubisco or by itself. The biochemical and structural experiments are well designed and clearly presented in the manuscript. Not only did they address the critical interaction between M58 and βCA at the atomic levels, they also revealed two unexpected interactions: one between the SSUL modules and γ-CAL domain of M58 and the headtohead interaction between γ-CAL domains from two M58 trimers. All these interactions lead to intricate network of proteins influenced by redox conditions. Hence, this manuscript is an important step towards understanding the detailed interactions among proteins inside β-carboxysomes and should be of great interest to the researchers studying β-carboxysomes as well as implementing them into plants for bioengineering purposes. Despite the intricate nature of the materials, the manuscript is well organized, and the findings are clearly described.
We thank the reviewer for her/his positive remarks.
Minor suggestions for the authors: 1. In line 202, when homo-demixing of M58 in Fig. 4c,d was described, it would be helpful to point out the difference between the experiments in Fig. 4c,d and those in Fig. 1b,c where M58 alone was shown not to form condensate.
We have clarified this point. Fig 8a ii, shouldn't the γCAL-CcaA condensate be salt "insensitive" based on Fig 2e? We apologize for the confusion. In the case of the γCAL-CcaA interaction, increasing salt concentration enhances the interaction, consistent with the contribution of hydrophobic forces to binding. To distinguish between the different salt sensitivities, we introduced arrows down and up in the figure to indicate reduced and enhanced interactions, respectively.

In
3. The paper from Gonzalez-Esquer et al (Streamlined construction of the cyanobacterial CO2-fixing organelle via protein domain fusions for use in plant synthetic biology, The Plant Cell, 27, 2637Cell, 27, -2644Cell, 27, , 2015 previously showed that a single chimeric protein was able to structurally and functionally replace four proteins: M58, M35, βCA and CcmN. The chimeric protein was derived by replacing γCAL in M58 with a shorter version of βCA. It was not understood how the chimeric protein was able to replace both M58 and M35. Would the new findings in the current manuscript somehow provide some 'speculative' explanation regarding the unusual behavior of that chimeric protein? For example, the current manuscript suggests there is differential redox-regulation of the interactions between Rubisco and M58 versus M35. More specifically, the interaction between SSUL modules and γCAL in M58, which appeared enhanced under oxidizing conditions, is no longer present with the chimeric protein. Could the redox regulation of the chimeric protein be more similar to M35, while at the same time, it is more structurally and physically similar to M58? Can the authors provide some insights into the chimeric protein in Gonzalez-Esquer et al paper in the discussion section? The results of Gonzalez-Esquer et al. are consistent with our findings. Assuming the βCA domain forms a tetramer as does CcaA in our study, the number of SSUL modules in the fusion protein (CcmC) would be higher (12) than in M58 (9). This might compensate for the contribution of γCAL to condensate formation. As the same time, since the βCA domain provides the carbonic anhydrase activity, recruitment of CccA via γCAL is no longer needed. Moreover, by linking the encapsulation peptide of CcmN to βCA, CcmN (which normally interacts with the γCAL domains of M58) is also no longer needed for carboxysome formation. However, with this construct the redox-regulation of carboxysome biogenesis/function is probably disturbed, as evident by the effect on the abundance, size and shape of βcarboxysomes in CcmC expressing cells. While we thank the reviewer for bringing this interesting paper to our attention, we feel that discussing this work would require extensive explanation of the interaction of CcmN with shell proteins. Given the space limitations, we defer a discussion of the Gonzalez-Esquer et al. paper to a future study. However, the paper is now cited in a more general context in the Introduction.

Reviewer #3 (plant structural biology)
Remarks to the Author: Carboxysome is a special microcompartment formed in cyanobacteria and algae that enclose the enzymes Rubisco and carbonic anhydrase to optimize photosynthetic carbon fixation. Understanding the biogenesis of carboxysome assembly and implementing an optimized carboxysome-like CO2 concentrating mechanism (CCM) into plants is a desirable strategy to increase the crop yield. In this study, the authors revealed a central role of the scaffolding protein CcmM M58 via initiating phase separation in the biogenesis of β-carboxysomes in cyanobacteria. Specially, the N-terminal γCAL domain of M58 mediate interactions with CcaA (support the CA activity), intermolecular interactions with the SSUL domain, and also head-to-head association of CcmM trimers. Overall, this study provides new sights into the biogenesis of β-carboxysome. I have a few concerns on the manuscript.
We thank the reviewer for critically reading the manuscript and her/his remarks.
1. In figure 2f and 3, it seems that W257 in the C2 peptide play crucial roles in mediating the interactions with the γCAL domain of M58. Further biochemical assays should be carried out to validate the structural model as well as the key involved residues.
We agree and have generated single point mutations W257A and R265D in both EGFP-C17 and CcaA to validate the structural model. Mutating Trp257 to Ala and Arg265 to Asp in the C2 sequence of CcaA reduced condensate formation with M58 by ~50%, or completely abolished condensate formation, respectively, as analyzed in the turbidity assay (see new Fig. 3d). Both mutations in EGFP-C17 abolished complex formation with γCAL by gel-shift assay (see new Supplementary Fig. 3i). These results underscore the specific contribution of hydrophobic and charge interactions to binding as indicated by the crystal structure.
2. CcaA exhibits as a tetramer in solution (Fig S1). It seems the preceding C1 peptide mediates the oligmerization. In the complex structure of γCAL-C2, each protomer of γCAL binds a C2 peptide. The conclusion "each protomer of the γCAL trimer interacts with one protomer of a CcaA tetramer" should be biochemically validated using full length proteins.
Deletion of more than 60 residues from the C-terminus of CcaA of Synechocystis PCC6803 has been shown to disrupt oligomer formation and enzymatic acitivity (So AK-C et al., Funct. Plant Biol. 2002). We have not independently validated the role of the C1 sequence in oligomerization. However, we now generated a construct in which the flexible C1 sequence, residues 205 to 219, were deleted and replaced by the sequence GSGGS in the CcaA protein of Se7942. This construct was still oligomeric by SECMALS and formed a condensate with M58 with wild-type efficiency, thus confirming the role of C2 as recognition sequence for binding to the γCAL domains. The role of C1 in oligomer formation would need to be investigated in the future. Note that the C1 sequence is not conserved between Se7942 and Synechocystis PCC6803.
The reviewer is correct in that we formerly do not know whether each γCAL protomer binds one CcaA tetramer. We only know that three monomers of EGFP-C217 can bind simultaneously ( Supplementary  Fig. 3h). However, for steric reasons, three CcaA tetramers may not be bound simultaneously, keeping in mind that these interactions are fluctuating. We apologize for the misleading statement, which we have changed to: "In summary, assuming the absence of steric hindrance, the γCAL trimer in the context of M58 may interact with 2 or 3 CcaA tetramers via the C2 sequence as the basis for condensate formation". Fig 6, for the 3.6 Å resolution map, the color gradient of the local resolution maps of side and end views from blue to red indicates local resolution from 2.0 to 6.0 Å. I suggest the authors show the values in a shorter range (3.0 to 6.0 Å?).

In Supplemental
We have made the requested change. Supplemental Fig 8, the authors showed the structural model of M58-RbcL8 to further validate the interaction between M58 and Rubisco complex. As the resolution of M58-RbcL8 is only 8 Å, information provided from the model is very limited and not reliable. I think this part is not necessary in the current manuscript.

In
We respectfully disagree. For a map with 8 Å resolution, the observation of the presence or absence of the SSUL module (11 KDa) is still reliable. Please note that to validate the map, we performed two rounds of 3D classification focusing on either the SSUL or the RbcS position on the RbcL2 unit ( Supplementary Fig. 8f). We consider this finding important evidence that SSUL cannot replace RbcS, and thus would like to maintain the data in the manuscript. Table 3, the authors should provide more parameters to show the model validations.

In supplementary
Please note that for all the reported cryo-EM structures no models were generated, and thus no model validations are required. Due to the low resolution of the EM maps, we docked the published PDB crystal structures into the EM density maps. The PDB codes mentioned in Table 3 were used to generate an initial 3D EM map for 3D classification during EM data processing.
6. In figures 1, 4, 7 and supplementary figs 2, 4, 10, the authors show the condensates of different proteins. In some figures, the authors exhibited the FRAP assays. It will be better to additionally show the results of droplet fusion.
As suggested, we analyzed droplet fusion for the various condensates. We find that the M58-CcaA and M58-M58 condensates do not undergo fusion during the 20 min observation period. Droplet fusion of M58 and Rubisco is observed, and the rate is very similar to that previously observed for M35-Rubisco droplets (Wang H et  7. The authors demonstrated that Rubisco, M58, M35 and CcaA could co-assembly in vitro. How about in vivo? It will be better to confirm the co-assembly in vivo. Previous in vivo analyses have clearly shown that Rubisco, M58 , M35 and CcaA are colocalized within carboxysomes (Long BM et al., J. Biol. Chem. 2007;Long BM et al., Plant Physiol. 2010;Niederhuber MJ et al., Mol. Biol. Cell 2017), and all 4 proteins can be pulled-down using N-terminally His-tagged M58. Unfortunately, it has not been possible to date to capture pre-carboxysomes and demonstrate the coassembly of the various proteins in vivo. Demonstration of such co-assembly in intact cyanobacterial cells will require a major effort, which is beyond the scope of this study.
8. In figure 6, the authors demonstrated that trimeric M58 binds rubisco with high affinity in a turbidity assay. Maybe some additional assays such as ITC will be better.
Unfortunately, ITC measurements of condensate forming mixtures are not possible as they result in clogging of the ITC chamber. Moreover, the absence of a defined stoichiometry would make any interpretation of such data difficult. Importantly, we compare the apparent M58-Rubisco affinity measured by the turbidity assay in Fig. 6a with our previous measurements for the M35-Rubisco interaction (Wang H et al., Nature 2019) using the same method.
9. CcmM directs multiprotein phase separation in β-carboxysome biogenesis. How about the effect of this phase separation on carbon fixation? Whether the ability of β-carboxysome carbon fixation depends on this phase separation?
Condensate formation of Rubisco, CcaA and other proteins, involved in carbon fixation, is the first step in the biogenesis of carboxysomes, and as such is required for the formation of a functional CCM. This condensate formation is mediated by the scaffolding protein CcmM (M58 and M35 in Synechococcus elongatus PCC 7942). However, once the carboxysome proteinaneous shell has formed, the material property of the condensate is most likely no longer critical to maintain the compartment. Thus, metabolic compartments, including carboxysomes, would differ from membraneless condensates, such as stress granules or the nucleolus, which do not have a shell and are dissociable. In fact, some of the multivalent interactions in the pre-carboxysome condensate are weakened (Wang H et al., Nature 2019) after shell formation, as the interior of the carboxysome is oxidizing (Price GD et al., Plant Physiol. 1992;Peña KL et al., PNAS 2010;Chen AH et al., PLoS One 2013). These aspects are now discussed more clearly in the revised manuscript.

Decision Letter, first revision: 16th Sep 2021
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Please use the following link for uploading these materials: [REDACTED] If you have any further questions, please feel free to contact me. Reviewer #1: Remarks to the Author: The authors have addressed most of my comments , but still fail to provide evidence for the roles of disordered regions, and establish connections with multivalent interactions. I think they still miss a key point for how condensates are formed, it is a pity that they have refused to perform at least some kind of bioinformatics predictions. Even if the key interaction is structured and specific, high entropy has to be achieved somehow. point for how condensates are formed, it is a pity that they have refused to perform at least some kind of bioinformatics predictions. Even if the key interaction is structured and specific, high entropy has to be achieved somehow.
We appreciate the opportunity to again respond to the comments of Reviewer #1. We would like to note that the primary focus of our manuscript is to understand the mechanism of carboxysome biogenesis. The principles of condensate formation have been discussed in several excellent reviews, cited in the manuscript. We agree with the reviewer that the flexible regions linking the SSUL domains play an important role: by joining the SSUL modules, they generate the critical multivalency for the interactions of SSUL with Rubisco and the γCAL domains of M58. Furthermore, their dynamic nature likely helps to balance the entropy penalty that occurs upon SSUL binding to Rubisco or γCAL. We have added the following sentence to that effect in the discussion: "The flexible linkers between the SSUL modules apparently do not contribute directly to the interaction, but may play a role in balancing the entropic penalty of SSUL binding." Reviewer #2 (photosynthesis, carboxysomes) The authors have sufficiently addressed the concerns in the revised manuscript and added some additional experimental data to show the importance of the W257 and R265 residues of the C2 peptide from CcaA on its interaction with γCAL. Through multiple biochemical and biophysical approaches as well as structural studies, the authors have identified and characterized the nature of interactions underpinning the association of CcaA and Rubisco with the central organizing protein, M58. These interactions are critical to understand the synthesis of carboxysomes. I have the following minor comments.
We thank the reviewer for critically reading the revised manuscript.
2. Fig. 2f, why did adding such short peptides as C2(15) and C2(17) to E-GFP change its mobility on the Native PAGE so dramatically? Whatever is slowing down its mobility appeared to be gone in R265D mutant of the C2(17) as can be seen in the Supplementary Fig. 3i.
Protein mobility on native-PAGE is determined by the size, shape and charge of a protein. Accordingly, size markers are no reliable indicator of migration. By attaching the C217 peptide (one negative and two positive charges) to EGFP, we reduced the negative net charge of EGFP from -8 to -7. In addition, we changed the shape of the molecule, assuming that the C2 peptide represents a dynamically attached moiety that increases the hydrodynamic radius. Both changes are expected to slow migration on nativePAGE, consistent with the experimental result. Note that SEC-MALS analysis showed that all the EGFP fusion constructs are monomeric in solution. The single charge mutant R265D in the C2 peptide increased the negative net charge of the protein to -9, i.e. more negative than EGFP alone (-8). Thus, assuming that the additional negative charge partially compensates for the effect of the more expanded shape of the fusion protein compared to EGFP, the mutant would be expected to migrate similar to EGFP.
3. Line 758, the name of the chemical, possibly KCl, in front of 100 mM is missing.? Corrected.
4. In Rubisco activity assay, please confirm that RuBP was present in excess in the reactions and was not rate-limiting. Based on my calculations, the reactions included 0.2 nmol of Rubisco active sites and 300 nmol of RuBP. If each Rubisco active site carboxylated 5 RuBP per second in the experiment, which is quite possible for the cyanoabacterial Rubisco at dissolved CO2 around its Km, all RuBP would be carboxylated in about 5 minutes. If the reactions were carried out under aerobic conditions, some RuBP would be oxygenated as well. Based on mixing 250 mM NaHCO3 and 4.5 mM NaH14CO3 with specific activity of 56.6 mCi/mmol), the amount of 14C activity counted after ending each reaction can be converted to mmol using 1 mCi/mmol. That amount should be well below 300 nmol to be able to compare the Rubisco activities across samples.
We appreciate the reviewer's concern. As suggested, we have performed the calculation using the 14C activity counts obtained after 5 min for Rubisco (RbcL8S8) on its own under both reducing and oxidizing conditions. In the presence of DTT we find a 14C activity of 152 nmol and of 158 nmol under oxidizing condition -well below 300 nmol. We conclude that the concentration of RuBP is not rate-limiting in our assays and that the Rubisco activities can be compared across the samples. The cpm data of the measurements are included in the source data file. Acceptance is conditional on the manuscript's not being published elsewhere and on there being no announcement of this work to the newspapers, magazines, radio or television until the publication date in Nature Structural & Molecular Biology.
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