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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.


Expression of Hnea rCsoS2 in E. coli with an N- or a C-terminal tag. (a) Schematic of the short and long form of rCsoS2 proteins produced by E. coli when codons for either an N- or a C-terminal tag are genetically fused to the open reading frame of Hnea csoS2 gene. In the case of a C-terminal tag, the short form (boxed by dotted lines) cannot be purified via affinity chromatography because of lack of the C-terminal tag. (b) Purified rCsoS2 in comparison with CsoS2A and CsoS2B from native source. The left lane shows the short (CsoS2A) and long (CsoS2B) form of CsoS2 protein in purified Hnea carboxysomes. When expressed in E. coli with an N-terminal tag, both the short and long forms can be purified using an affinity column (middle lane). When expressed in E. coli with a C-terminal tag, only the long form can be recovered after affinity purification followed by self-cleavage of the tag.
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life-05-01141-f002: Expression of Hnea rCsoS2 in E. coli with an N- or a C-terminal tag. (a) Schematic of the short and long form of rCsoS2 proteins produced by E. coli when codons for either an N- or a C-terminal tag are genetically fused to the open reading frame of Hnea csoS2 gene. In the case of a C-terminal tag, the short form (boxed by dotted lines) cannot be purified via affinity chromatography because of lack of the C-terminal tag. (b) Purified rCsoS2 in comparison with CsoS2A and CsoS2B from native source. The left lane shows the short (CsoS2A) and long (CsoS2B) form of CsoS2 protein in purified Hnea carboxysomes. When expressed in E. coli with an N-terminal tag, both the short and long forms can be purified using an affinity column (middle lane). When expressed in E. coli with a C-terminal tag, only the long form can be recovered after affinity purification followed by self-cleavage of the tag.

Mentions: To test if the shorter form of Hnea CsoS2 is a result of post-translation processing on the C-terminus of the full-length protein, we heterogeneously expressed the Hnea csoS2 gene in E. coli with either an N- or a C-terminal tag. Two forms of the N-terminally tagged CsoS2 can be purified by affinity chromatography, and the short form is more abundant (Figure 2). This may be due to different susceptibilities to proteolysis in E. coli, or indicate that most of the N-terminally His-tagged rCsoS2 is expressed as a short form (CsoS2A) with an intact N-terminus. In contrast, only the long form (CsoS2B) was eluted from the affinity resin after self-cleavage of the C-terminal intein tag (Figure 2). Collectively, these findings confirm that CsoS2B is the full-length polypeptide while CsoS2A is C-terminally truncated. The mechanism of truncation is self-contained in the Hnea csoS2 gene regardless of expression host. In an attempt to identify the C-terminal truncation site, matrix-assisted laser desorption/ionization time-of-light mass spectrometer (MALDI-TOF MS) was used to analyze in-gel Trypsin digested CsoS2A and CsoS2B from purified Hnea carboxysomes (Figure S1). The coverage of full-length CsoS2B is 44% and the last detectable peptide covers the sequence up to R868, which is the penultimate residue (Figure S1b). The last detectable peptide of CsoS2A covers the sequence up to R836 (Figure S1a). However, a truncation of N837-G869 will only result in a MW difference of 3.3 kDa; the observed MW difference between CsoS2A and CsoS2B is 45 kDa [6]. Close inspection of the MALDI-TOF results for CsoS2A reveal that there is no coverage over a large region (G574–R826) in contrast to ample coverage in the CsoS2B sample in the same region. Therefore, the coverage between V827–R836 may be an aberration. The last detected residue in CsoS2A prior to this region is R573. A truncated CsoS2 protein with residues 1-573 will yield a calculated MW of 61 kDa, which is 31 kDa smaller than the calculated MW of the full-length CsoS2. This calculated 31 kDa difference is much closer to the observed MW difference of 45 kDa.


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)

Expression of Hnea rCsoS2 in E. coli with an N- or a C-terminal tag. (a) Schematic of the short and long form of rCsoS2 proteins produced by E. coli when codons for either an N- or a C-terminal tag are genetically fused to the open reading frame of Hnea csoS2 gene. In the case of a C-terminal tag, the short form (boxed by dotted lines) cannot be purified via affinity chromatography because of lack of the C-terminal tag. (b) Purified rCsoS2 in comparison with CsoS2A and CsoS2B from native source. The left lane shows the short (CsoS2A) and long (CsoS2B) form of CsoS2 protein in purified Hnea carboxysomes. When expressed in E. coli with an N-terminal tag, both the short and long forms can be purified using an affinity column (middle lane). When expressed in E. coli with a C-terminal tag, only the long form can be recovered after affinity purification followed by self-cleavage of the tag.
© Copyright Policy
Related In: Results  -  Collection

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

life-05-01141-f002: Expression of Hnea rCsoS2 in E. coli with an N- or a C-terminal tag. (a) Schematic of the short and long form of rCsoS2 proteins produced by E. coli when codons for either an N- or a C-terminal tag are genetically fused to the open reading frame of Hnea csoS2 gene. In the case of a C-terminal tag, the short form (boxed by dotted lines) cannot be purified via affinity chromatography because of lack of the C-terminal tag. (b) Purified rCsoS2 in comparison with CsoS2A and CsoS2B from native source. The left lane shows the short (CsoS2A) and long (CsoS2B) form of CsoS2 protein in purified Hnea carboxysomes. When expressed in E. coli with an N-terminal tag, both the short and long forms can be purified using an affinity column (middle lane). When expressed in E. coli with a C-terminal tag, only the long form can be recovered after affinity purification followed by self-cleavage of the tag.
Mentions: To test if the shorter form of Hnea CsoS2 is a result of post-translation processing on the C-terminus of the full-length protein, we heterogeneously expressed the Hnea csoS2 gene in E. coli with either an N- or a C-terminal tag. Two forms of the N-terminally tagged CsoS2 can be purified by affinity chromatography, and the short form is more abundant (Figure 2). This may be due to different susceptibilities to proteolysis in E. coli, or indicate that most of the N-terminally His-tagged rCsoS2 is expressed as a short form (CsoS2A) with an intact N-terminus. In contrast, only the long form (CsoS2B) was eluted from the affinity resin after self-cleavage of the C-terminal intein tag (Figure 2). Collectively, these findings confirm that CsoS2B is the full-length polypeptide while CsoS2A is C-terminally truncated. The mechanism of truncation is self-contained in the Hnea csoS2 gene regardless of expression host. In an attempt to identify the C-terminal truncation site, matrix-assisted laser desorption/ionization time-of-light mass spectrometer (MALDI-TOF MS) was used to analyze in-gel Trypsin digested CsoS2A and CsoS2B from purified Hnea carboxysomes (Figure S1). The coverage of full-length CsoS2B is 44% and the last detectable peptide covers the sequence up to R868, which is the penultimate residue (Figure S1b). The last detectable peptide of CsoS2A covers the sequence up to R836 (Figure S1a). However, a truncation of N837-G869 will only result in a MW difference of 3.3 kDa; the observed MW difference between CsoS2A and CsoS2B is 45 kDa [6]. Close inspection of the MALDI-TOF results for CsoS2A reveal that there is no coverage over a large region (G574–R826) in contrast to ample coverage in the CsoS2B sample in the same region. Therefore, the coverage between V827–R836 may be an aberration. The last detected residue in CsoS2A prior to this region is R573. A truncated CsoS2 protein with residues 1-573 will yield a calculated MW of 61 kDa, which is 31 kDa smaller than the calculated MW of the full-length CsoS2. This calculated 31 kDa difference is much closer to the observed MW difference of 45 kDa.

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.