A β-type carbonic anhydrase, PtCA1, of the marine diatom Phaeodactylum tricornutum was previously shown to be present in the chloroplast as clumped particles on the girdle lamellae. A series of deletions was carried out on the PtCA1 gene, ptca1, at regions encoding N- or C-terminal domains of the mature PtCA1. These deletion constructs were fused with the EGFP [enhanced GFP (green fluorescent protein)] gene, egfp, introduced and expressed in the cells of P. tricornutum. All three types of N-terminal deletions, Δ52–63, Δ64–75 and Δ76–87 relative to the initiation methionine, showed little interference with the particle formation of the PtCA1::GFP fusion protein. Similarly, one of the three types of C-terminal deletions, Δ253–262, was silent. However, the remaining two C-terminal deletions, Δ263–272 and Δ273–282 relative to the initiation methionine, were strongly inhibitory to the particle formation of PtCA1. The C-terminal 263–282 region comprises five hydrophobic amino acids, Met263, Leu266, Ile269, Leu272 and Leu275, which were predicted to form a hydrophobic cluster on the C-terminal α-helix. Each or all five of these hydrophobic residues were replaced with a hydrophilic residue with a side chain of similar size and structure, glutamate. Particle formations of PtCA1 were moderately inhibited by substitutions of Met263, Leu266 and Ile269 but more evidently by substitutions of Leu272 and Leu275. Finally, substitutions of all five hydrophobic residues resulted in an efficient inhibition of particle formation and the GFP signal was totally dispersed throughout the stroma area. These results strongly suggest that the amphipathic C-terminal helix of PtCA1 plays an essential role in the formation of the macromolecular protein complex.
Formation of macromolecular complexes of carbonic anhydrases in the chloroplast of a marine diatom by the action of the C-terminal helix
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Yoshiko Kitao, Yusuke Matsuda; Formation of macromolecular complexes of carbonic anhydrases in the chloroplast of a marine diatom by the action of the C-terminal helix. Biochem J 1 May 2009; 419 (3): 681–688. doi: https://doi.org/10.1042/BJ20082315
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