Limits...
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.


Pull-down assay and purification of α-carboxysome interaction complexes. (a) SDS-PAGE and Western blots (α-CsoS1 and α-CsoS1D) of lysate (L) from cells co-expressing CsoS1 and CsoS2 or CsoS1, CsoS1D and CsoS2 and the corresponding cell debris after breaking the cells (P), flow-through fractions (FT) and elutions (E) after pull-down assay using Glutathione-Sepharose magnetic beads; (b) SDS-PAGE of lysate (L) from cells co-expressing CsoS1, CsoS1D and CsoS2 from a single construct, the isolated complex (I) and the elution (E) from pull-down assay using Ni-NTA-agarose magnetic beads. Western blots for samples L and I are shown underneath using four different antibodies: α-His5, α-CsoS2-C, α-CsoS1 and α-CsoS1D. All three bands that have higher MW (between 75–100 kDa) are identified as CsoS2 with intact C-termini.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4499774&req=5

life-05-01141-f011: Pull-down assay and purification of α-carboxysome interaction complexes. (a) SDS-PAGE and Western blots (α-CsoS1 and α-CsoS1D) of lysate (L) from cells co-expressing CsoS1 and CsoS2 or CsoS1, CsoS1D and CsoS2 and the corresponding cell debris after breaking the cells (P), flow-through fractions (FT) and elutions (E) after pull-down assay using Glutathione-Sepharose magnetic beads; (b) SDS-PAGE of lysate (L) from cells co-expressing CsoS1, CsoS1D and CsoS2 from a single construct, the isolated complex (I) and the elution (E) from pull-down assay using Ni-NTA-agarose magnetic beads. Western blots for samples L and I are shown underneath using four different antibodies: α-His5, α-CsoS2-C, α-CsoS1 and α-CsoS1D. All three bands that have higher MW (between 75–100 kDa) are identified as CsoS2 with intact C-termini.

Mentions: The protein precipitates frequently formed by rCsoS2 and rCsoS2 upon interaction with other carboxysome components in vitro in protein-protein interaction studies make it difficult to isolate complexes of defined stoichiometry. Therefore, co-expression of potential binding partners and isolation of interaction complexes were pursued. GST-MED4 CsoS2 (pFC005) was co-expressed with CsoS1 alone (pFC117) or CsoS1 and CsoS1D (pFC119) (vector information see Table S3). Pull-down experiments were performed for both. Although no CsoS1 was found in elution fractions examined by coomassie blue stained SDS-PAGE, when CsoS1D is also co-expressed, CsoS1 can be detected in the elution fraction by Western Blotting (Figure 11a). CsoS1D is present at approximately a 5:1 molar ratio to GST-CsoS2 based on densitometry of stained SDS-PAGE (Figure 11a, fraction E). In contrast, the estimated copy number of CsoS1D:CsoS2 is 1:4 based on analysis of purified MED4 carboxysomes [8]. Since stoichiometry is likely a factor for proper complex assembly, we next varied protein expression levels using E. coli RBS with different strengths. A single-plasmid co-expression construct, pFC215, was built with codon-optimized csoS1, csoS1D and csoS2 genes and artificial intergenic regions. CsoS1-CsoS1D-CsoS2 complexes were purified following a protocol modified and adapted from both α-carboxysome purification and synthetic rBMC shell purification protocols [2,8]. Interestingly, all three high MW polypeptides present in the purified sample are identified as CsoS2 with intact C-termini (Figure 11b). Pull-down experiments were also performed for this expression construct, and the protein composition of the pulled-down fraction is similar to that of purified complex (Figure 11b).


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)

Pull-down assay and purification of α-carboxysome interaction complexes. (a) SDS-PAGE and Western blots (α-CsoS1 and α-CsoS1D) of lysate (L) from cells co-expressing CsoS1 and CsoS2 or CsoS1, CsoS1D and CsoS2 and the corresponding cell debris after breaking the cells (P), flow-through fractions (FT) and elutions (E) after pull-down assay using Glutathione-Sepharose magnetic beads; (b) SDS-PAGE of lysate (L) from cells co-expressing CsoS1, CsoS1D and CsoS2 from a single construct, the isolated complex (I) and the elution (E) from pull-down assay using Ni-NTA-agarose magnetic beads. Western blots for samples L and I are shown underneath using four different antibodies: α-His5, α-CsoS2-C, α-CsoS1 and α-CsoS1D. All three bands that have higher MW (between 75–100 kDa) are identified as CsoS2 with intact C-termini.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-01141-f011: Pull-down assay and purification of α-carboxysome interaction complexes. (a) SDS-PAGE and Western blots (α-CsoS1 and α-CsoS1D) of lysate (L) from cells co-expressing CsoS1 and CsoS2 or CsoS1, CsoS1D and CsoS2 and the corresponding cell debris after breaking the cells (P), flow-through fractions (FT) and elutions (E) after pull-down assay using Glutathione-Sepharose magnetic beads; (b) SDS-PAGE of lysate (L) from cells co-expressing CsoS1, CsoS1D and CsoS2 from a single construct, the isolated complex (I) and the elution (E) from pull-down assay using Ni-NTA-agarose magnetic beads. Western blots for samples L and I are shown underneath using four different antibodies: α-His5, α-CsoS2-C, α-CsoS1 and α-CsoS1D. All three bands that have higher MW (between 75–100 kDa) are identified as CsoS2 with intact C-termini.
Mentions: The protein precipitates frequently formed by rCsoS2 and rCsoS2 upon interaction with other carboxysome components in vitro in protein-protein interaction studies make it difficult to isolate complexes of defined stoichiometry. Therefore, co-expression of potential binding partners and isolation of interaction complexes were pursued. GST-MED4 CsoS2 (pFC005) was co-expressed with CsoS1 alone (pFC117) or CsoS1 and CsoS1D (pFC119) (vector information see Table S3). Pull-down experiments were performed for both. Although no CsoS1 was found in elution fractions examined by coomassie blue stained SDS-PAGE, when CsoS1D is also co-expressed, CsoS1 can be detected in the elution fraction by Western Blotting (Figure 11a). CsoS1D is present at approximately a 5:1 molar ratio to GST-CsoS2 based on densitometry of stained SDS-PAGE (Figure 11a, fraction E). In contrast, the estimated copy number of CsoS1D:CsoS2 is 1:4 based on analysis of purified MED4 carboxysomes [8]. Since stoichiometry is likely a factor for proper complex assembly, we next varied protein expression levels using E. coli RBS with different strengths. A single-plasmid co-expression construct, pFC215, was built with codon-optimized csoS1, csoS1D and csoS2 genes and artificial intergenic regions. CsoS1-CsoS1D-CsoS2 complexes were purified following a protocol modified and adapted from both α-carboxysome purification and synthetic rBMC shell purification protocols [2,8]. Interestingly, all three high MW polypeptides present in the purified sample are identified as CsoS2 with intact C-termini (Figure 11b). Pull-down experiments were also performed for this expression construct, and the protein composition of the pulled-down fraction is similar to that of purified complex (Figure 11b).

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.