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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.


Related in: MedlinePlus

A Hnea mutant with SPA-tagged CsoS2. (a) The SPA tag fused to the C-terminus of CsoS2 contains a 3x FLAG epitope and a calmodulin binding domain (CBD) separated by a TEV protease site. A kanamycin resistance gene (KmR) cassette follows the SPA tag to allow for selection. (b) Both wildtype and mutant carboxysomes can be purified and their polypeptide separation patterns are similar, except SPA-tagged CsoS2B is slightly larger than untagged CsoS2B. (c) Western blots of wildtype and mutant cells blocked against α-CsoS2 antisera and α-FLAG antibodies. Only the long form of CsoS2 has a FLAG epitope tag in HnSPAS2. No cross-reactivity with small polypeptides was observed. (d) Purified wildtype and HnSPAS2 mutant carboxysomes are indistinguishable in TEM images.
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life-05-01141-f004: A Hnea mutant with SPA-tagged CsoS2. (a) The SPA tag fused to the C-terminus of CsoS2 contains a 3x FLAG epitope and a calmodulin binding domain (CBD) separated by a TEV protease site. A kanamycin resistance gene (KmR) cassette follows the SPA tag to allow for selection. (b) Both wildtype and mutant carboxysomes can be purified and their polypeptide separation patterns are similar, except SPA-tagged CsoS2B is slightly larger than untagged CsoS2B. (c) Western blots of wildtype and mutant cells blocked against α-CsoS2 antisera and α-FLAG antibodies. Only the long form of CsoS2 has a FLAG epitope tag in HnSPAS2. No cross-reactivity with small polypeptides was observed. (d) Purified wildtype and HnSPAS2 mutant carboxysomes are indistinguishable in TEM images.

Mentions: A sequential peptide affinity (SPA) tag that includes a calmodulin binding domain (CBD), a tobacco etch virus (TEV) protease recognition site and three copies of the FLAG epitope, was fused to the C-terminus of Hnea CsoS2 to generate the HnSPAS2 mutant (Figure 4a). This mutant is able to grow in ambient CO2, and HnSPAS2 mutant carboxysomes can be purified using the standard protocols [26]. Transmission electron microscopy revealed that the size and shape of HnSPAS2 mutant carboxysomes are indistinguishable from their wildtype counterparts (Figure 4d). The composition of purified HnSPAS2 mutant carboxysomes was analyzed by SDS-PAGE, revealing a similar pattern of polypeptide migration compared to wildtype Hnea carboxysomes, with the exception of the C-terminal SPA tagged CsoS2B. The full-length CsoS2 polypeptide migrates more slowly than untagged CsoS2B, as would be expected from the addition of the 7.7 kDa SPA tag (Figure 4b). Cell extracts of wildtype Hnea and two HnSPAS2 mutant clones were subjected to immunoblotting (Figure 4c). Probing with α-HneaCsoS2 antisera showed that both short form and long form of CsoS2 are present in the HnSPAS2 mutant, as expected. Probing with α-FLAG antibodies revealed that only the long form contains the FLAG epitope tag. No cross-reactivity of the α-HneaCsoS2 antisera or α-FLAG antibodies with any small polypeptides was observed. These findings confirmed that the long form, CsoS2B, is the full length CsoS2 protein. Interestingly, purified HnSPAS2 mutant carboxysomes can bind to agarose beads conjugated with α-FLAG antibodies and elute with 100 mM glycine buffer, pH 2.5. In contrast, no detectable amount of wildtype carboxysomes is recovered in a comparable pull-down experiment (Figure S3). Densitometric analysis revealed that the mass ratio of polypeptides in the eluted HnSPAS2 carboxysomes is 2:1.5:1:11:1.5:7 for CsoS2B:CsoS2A:CsoSCA:L8S8RuBisCO:CsoS1B:CsoS1A/C, (L8S8RuBisCO consists of eight polypeptides each of large (CbbL) and small (CbbS) subunits). These values are similar to those of purified intact HnSPAS2 carboxysomes (CsoS2B:CsoS2A:CsoSCA:L8S8RuBisCO:CsoS1B:CsoS1A/C = 2.5:2.0:1:13:1:6), suggesting that the trapped carboxysomes were intact.


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 Hnea mutant with SPA-tagged CsoS2. (a) The SPA tag fused to the C-terminus of CsoS2 contains a 3x FLAG epitope and a calmodulin binding domain (CBD) separated by a TEV protease site. A kanamycin resistance gene (KmR) cassette follows the SPA tag to allow for selection. (b) Both wildtype and mutant carboxysomes can be purified and their polypeptide separation patterns are similar, except SPA-tagged CsoS2B is slightly larger than untagged CsoS2B. (c) Western blots of wildtype and mutant cells blocked against α-CsoS2 antisera and α-FLAG antibodies. Only the long form of CsoS2 has a FLAG epitope tag in HnSPAS2. No cross-reactivity with small polypeptides was observed. (d) Purified wildtype and HnSPAS2 mutant carboxysomes are indistinguishable in TEM images.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-01141-f004: A Hnea mutant with SPA-tagged CsoS2. (a) The SPA tag fused to the C-terminus of CsoS2 contains a 3x FLAG epitope and a calmodulin binding domain (CBD) separated by a TEV protease site. A kanamycin resistance gene (KmR) cassette follows the SPA tag to allow for selection. (b) Both wildtype and mutant carboxysomes can be purified and their polypeptide separation patterns are similar, except SPA-tagged CsoS2B is slightly larger than untagged CsoS2B. (c) Western blots of wildtype and mutant cells blocked against α-CsoS2 antisera and α-FLAG antibodies. Only the long form of CsoS2 has a FLAG epitope tag in HnSPAS2. No cross-reactivity with small polypeptides was observed. (d) Purified wildtype and HnSPAS2 mutant carboxysomes are indistinguishable in TEM images.
Mentions: A sequential peptide affinity (SPA) tag that includes a calmodulin binding domain (CBD), a tobacco etch virus (TEV) protease recognition site and three copies of the FLAG epitope, was fused to the C-terminus of Hnea CsoS2 to generate the HnSPAS2 mutant (Figure 4a). This mutant is able to grow in ambient CO2, and HnSPAS2 mutant carboxysomes can be purified using the standard protocols [26]. Transmission electron microscopy revealed that the size and shape of HnSPAS2 mutant carboxysomes are indistinguishable from their wildtype counterparts (Figure 4d). The composition of purified HnSPAS2 mutant carboxysomes was analyzed by SDS-PAGE, revealing a similar pattern of polypeptide migration compared to wildtype Hnea carboxysomes, with the exception of the C-terminal SPA tagged CsoS2B. The full-length CsoS2 polypeptide migrates more slowly than untagged CsoS2B, as would be expected from the addition of the 7.7 kDa SPA tag (Figure 4b). Cell extracts of wildtype Hnea and two HnSPAS2 mutant clones were subjected to immunoblotting (Figure 4c). Probing with α-HneaCsoS2 antisera showed that both short form and long form of CsoS2 are present in the HnSPAS2 mutant, as expected. Probing with α-FLAG antibodies revealed that only the long form contains the FLAG epitope tag. No cross-reactivity of the α-HneaCsoS2 antisera or α-FLAG antibodies with any small polypeptides was observed. These findings confirmed that the long form, CsoS2B, is the full length CsoS2 protein. Interestingly, purified HnSPAS2 mutant carboxysomes can bind to agarose beads conjugated with α-FLAG antibodies and elute with 100 mM glycine buffer, pH 2.5. In contrast, no detectable amount of wildtype carboxysomes is recovered in a comparable pull-down experiment (Figure S3). Densitometric analysis revealed that the mass ratio of polypeptides in the eluted HnSPAS2 carboxysomes is 2:1.5:1:11:1.5:7 for CsoS2B:CsoS2A:CsoSCA:L8S8RuBisCO:CsoS1B:CsoS1A/C, (L8S8RuBisCO consists of eight polypeptides each of large (CbbL) and small (CbbS) subunits). These values are similar to those of purified intact HnSPAS2 carboxysomes (CsoS2B:CsoS2A:CsoSCA:L8S8RuBisCO:CsoS1B:CsoS1A/C = 2.5:2.0:1:13:1:6), suggesting that the trapped carboxysomes were intact.

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.


Related in: MedlinePlus