[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-562] Mechanisms for localization and location specific functions of bestrophin in the marine diatom Phaeodactylum tricornutum

○Karin Niwa¹, Minori Nigishi¹, Yusuke Matsuda¹ (1. Grad. Sch. Sci. Tech., Kwansei Gakuin Univ. (Japan))

Keywords:

Pyrenoid,Bestrophin,Diatom,Photosynthesis,CO2-concentrating mechanism

Seawater under the current atmospheric pCO₂ contains low dissolved CO₂ concentration which is far insufficient to saturate the CO₂ fixation enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The marine diatom, Phaeodactylum tricornutum possesses a CO₂-concentrating mechanism (CCM) that takes up external dissolved-inorganic carbon (DIC) and increases the CO₂ concentration around Rubisco. The pyrenoid in the chloroplast is a condensate of Rubisco formed by liquid–liquid phase separation. Thylakoid membranes penetrating the pyrenoid (pyrenoid-penetrating thylakoid; PPT) are the site where high concentrations of CO₂ are generated from accumulated stromal bicarbonate. Bestrophin (BST), transporters are proposed to supply HCO₃^- to the PPT lumen, and the HCO₃^- in the PPT lumen is rapidly dehydrated by a carbonic anhydrase specifically localized in the PPT lumen, generating an ample flux of CO₂ to Rubisco. The knockout (KO) strain of PtBST1, a stroma-thylakoidal HCO₃^- channel, showed decrease in DIC affinity and increase in NPQ, a thermal dissipation index of the photosystem II. Besides stroma-tylakoid BSTs, there is PtBST3, a PPT membrane factor, which possesses a unique C-terminal 113-amino-acid extension (113CST). When 113CST was deleted, the localization of PtBST3 changed from the vicinity of the pyrenoid to the entire stroma thylakoid, suggesting that the C-terminal stretch of PtBST3 is involved in PPT localization. A slight decrease in DIC affinity was detected in the PtBST3 KO strain, although the effect was much smaller than that of PtBST1 KO. When the 113CST of PtBST3 was tagged to GFP, the fusion protein was localized to the pyrenoid. In contrast, GFP tagging protein of PtBST3 with stepwise deleted 113CST showed stromal localization when more than 23 C-terminal residues were removed from 113CST. These results indicate that a pyrenoid-targeting sequence resides within the 22 residues (499–519) of PtBST3. When 113CST-deleted PtBST3 or the PtBST1:113CST:GFP fusion proteins were expressed in PtBST1- and PtBST3-KO strains, most transformants showed DIC affinity and NPQ regulation comparable to those of the wild type cells. However, in the PtBST3-KO strain expressing PtBST1:113CST:GFP, recovery of DIC affinity was observed, whereas NPQ was decreased. In this strain, fluorescence of PtBST1:113CST:GFP was observed surrounding the pyrenoid. These results strongly suggest that the PPT is mislocated in the PtBST3-KO strain.