Formation of macromolecular complexes of carbonic anhydrases in the chloroplast of a marine diatom by the action of the C-terminal helix.
A beta-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, Delta52-63, Delta64-75 and Delta76-87 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, Delta253-262, was silent. However, the remaining two C-terminal deletions, Delta263-272 and Delta273-282 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, Met(263), Leu(266), Ile(269), Leu(272) and Leu(275), which were predicted to form a hydrophobic cluster on the C-terminal alpha-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 Met(263), Leu(266) and Ile(269) but more evidently by substitutions of Leu(272) and Leu(275). 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.
Kitao
Yoshiko
Y
Department of Bioscience, School of Science and Technology, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo, Japan.
Matsuda
Yusuke
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D017377:Diatoms / Q000166:cytology / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012623:Seawater; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002256:Carbonic Anhydrases