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Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.


A working model for the location and function of CsoS2 in α-carboxysome assembly. Prior to α-carboxysome formation, RuBisCO and shell proteins such as CsoS1 are recruited by CsoS2. Subsequently, CsoS1 hexamers tile together and form shells anchored by CsoS2 via its C-region, and RuBisCO line up while associated with CsoS2. As a result, the carboxysome is assembled during the simultaneous formation of the shell and packing of RuBisCOs. CsoS2 may adapt different conformations in the final stage; a network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved Cysteine residues found in M-repeats. Short forms of CsoS2 (CsoS2A) will only organize RuBisCO but not provide anchoring to the shell. The tail of C-region may be exposed on the surface of the carboxysome and accessible from cytoplasm.
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life-05-01141-f012: A working model for the location and function of CsoS2 in α-carboxysome assembly. Prior to α-carboxysome formation, RuBisCO and shell proteins such as CsoS1 are recruited by CsoS2. Subsequently, CsoS1 hexamers tile together and form shells anchored by CsoS2 via its C-region, and RuBisCO line up while associated with CsoS2. As a result, the carboxysome is assembled during the simultaneous formation of the shell and packing of RuBisCOs. CsoS2 may adapt different conformations in the final stage; a network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved Cysteine residues found in M-repeats. Short forms of CsoS2 (CsoS2A) will only organize RuBisCO but not provide anchoring to the shell. The tail of C-region may be exposed on the surface of the carboxysome and accessible from cytoplasm.

Mentions: The results of our study suggest CsoS2 is highly flexible and that its three distinct regions have different binding specificities for RuBisCO and shell proteins. From these data and the results of prior studies, we propose a model for CsoS2 function and spatial location in α-carboxysomes (Figure 12). First, the N-region of CsoS2 recruits CsoS1 shell protein(s); when the local concentration increases to a threshold amount, CsoS1 starts to self-assemble into a single layer anchored upon the C-region of CsoS2 but leaving the tail of the C-region exposed to the cytoplasm. Simultaneously, RuBisCO coalesces with CsoS2 through protein-protein interactions, and a lattice of RuBisCO starts to form around the M-region while also simultaneously anchored to the C-region of CsoS2. As a result, RuBisCO is organized by the M-region of CsoS2. A network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved cysteine residues found in M-repeats. Carboxysomes of some species also have a short form of CsoS2, which is composed of only N- and M-regions. This form of CsoS2 would not be in contact with the shell but would be expected to organize the inner layers of RuBisCO only. In the absence of carboxysomal form IA RuBisCO, empty shells form, which contain both long and short form of CsoS2 due to the protein-protein interaction among CsoS2 proteins. This model is consistent with the majority of experimental observations. For example, in this model, only the outermost layer of RuBisCO can bind to the C-region of CsoS2 since CsoS2 is anchored on the shell via its C-region; RuBisCO of the inner layers interacts less strongly with flexible CsoS2 through the M-regions. This would explain the observation that the outmost layer of RuBisCO, which is closest to the shell, is the most ordered, while inner layers of RuBisCO may be a result of more random packing [44,45]. The model is also supported by the approximately 1/3 of carboxysomal RuBisCO that remains with the shell fraction when Hnea carboxysomes are disrupted by freeze-thawing; this RuBisCO cannot be released from the shell [50]. Although CsoS2 adopting a beads-on-a-string conformation for the M-region is not supported by solution state data on isolated CsoS2, it may still be the case in vivo; when it is packed with its interaction partner RuBisCO in a micro-environment where the local protein concentration is extremely high (approximately 900 mg/mL; see supporting methods for calculation). Furthermore, CsoS2 is predicted to be an intrinsically disordered protein (IDP) (Section 2.8, above); IDPs frequently adopt local folds specifically in the presence of interaction partners [51,52,53].


Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA - Life (Basel) (2015)

A working model for the location and function of CsoS2 in α-carboxysome assembly. Prior to α-carboxysome formation, RuBisCO and shell proteins such as CsoS1 are recruited by CsoS2. Subsequently, CsoS1 hexamers tile together and form shells anchored by CsoS2 via its C-region, and RuBisCO line up while associated with CsoS2. As a result, the carboxysome is assembled during the simultaneous formation of the shell and packing of RuBisCOs. CsoS2 may adapt different conformations in the final stage; a network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved Cysteine residues found in M-repeats. Short forms of CsoS2 (CsoS2A) will only organize RuBisCO but not provide anchoring to the shell. The tail of C-region may be exposed on the surface of the carboxysome and accessible from cytoplasm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499774&req=5

life-05-01141-f012: A working model for the location and function of CsoS2 in α-carboxysome assembly. Prior to α-carboxysome formation, RuBisCO and shell proteins such as CsoS1 are recruited by CsoS2. Subsequently, CsoS1 hexamers tile together and form shells anchored by CsoS2 via its C-region, and RuBisCO line up while associated with CsoS2. As a result, the carboxysome is assembled during the simultaneous formation of the shell and packing of RuBisCOs. CsoS2 may adapt different conformations in the final stage; a network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved Cysteine residues found in M-repeats. Short forms of CsoS2 (CsoS2A) will only organize RuBisCO but not provide anchoring to the shell. The tail of C-region may be exposed on the surface of the carboxysome and accessible from cytoplasm.
Mentions: The results of our study suggest CsoS2 is highly flexible and that its three distinct regions have different binding specificities for RuBisCO and shell proteins. From these data and the results of prior studies, we propose a model for CsoS2 function and spatial location in α-carboxysomes (Figure 12). First, the N-region of CsoS2 recruits CsoS1 shell protein(s); when the local concentration increases to a threshold amount, CsoS1 starts to self-assemble into a single layer anchored upon the C-region of CsoS2 but leaving the tail of the C-region exposed to the cytoplasm. Simultaneously, RuBisCO coalesces with CsoS2 through protein-protein interactions, and a lattice of RuBisCO starts to form around the M-region while also simultaneously anchored to the C-region of CsoS2. As a result, RuBisCO is organized by the M-region of CsoS2. A network of CsoS2 is formed based on inter-molecular interactions among CsoS2 proteins, which may be mediated through disulfide bonds formed between conserved cysteine residues found in M-repeats. Carboxysomes of some species also have a short form of CsoS2, which is composed of only N- and M-regions. This form of CsoS2 would not be in contact with the shell but would be expected to organize the inner layers of RuBisCO only. In the absence of carboxysomal form IA RuBisCO, empty shells form, which contain both long and short form of CsoS2 due to the protein-protein interaction among CsoS2 proteins. This model is consistent with the majority of experimental observations. For example, in this model, only the outermost layer of RuBisCO can bind to the C-region of CsoS2 since CsoS2 is anchored on the shell via its C-region; RuBisCO of the inner layers interacts less strongly with flexible CsoS2 through the M-regions. This would explain the observation that the outmost layer of RuBisCO, which is closest to the shell, is the most ordered, while inner layers of RuBisCO may be a result of more random packing [44,45]. The model is also supported by the approximately 1/3 of carboxysomal RuBisCO that remains with the shell fraction when Hnea carboxysomes are disrupted by freeze-thawing; this RuBisCO cannot be released from the shell [50]. Although CsoS2 adopting a beads-on-a-string conformation for the M-region is not supported by solution state data on isolated CsoS2, it may still be the case in vivo; when it is packed with its interaction partner RuBisCO in a micro-environment where the local protein concentration is extremely high (approximately 900 mg/mL; see supporting methods for calculation). Furthermore, CsoS2 is predicted to be an intrinsically disordered protein (IDP) (Section 2.8, above); IDPs frequently adopt local folds specifically in the presence of interaction partners [51,52,53].

Bottom Line: Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins.Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome.Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA. fcai@lbl.gov.

ABSTRACT
The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

No MeSH data available.