PARENT SESSION
Posters P4C Controling CO₂: Stomates and carbon concentrating mechanisms. Abstracts (631-642)

Sorting of -carbonic anhydrase into storomal particles in the marine diatom Phaeodactylum tricornutum. Daisuke Nakatsuma¹, Yusuke Matsuda^*,1, ¹ Department of Bioscience, Sanda, Hyogo, Japan

ABSTRACT- -carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum is encoded by nuclear genome. This enzyme was previously shown to be crucial for the operation of high-affinity photosynthesis for dissolved-inorganic carbon (DIC) and to be regulated at the transcriptional level upon changes in ambient [CO₂]. Both cDNA and protein of P. tricornutum CA (ptca and PtCA, respectively) were already isolated and cDNA sequence was shown to possess presequence of 138bp (pre138), which encodes 46 N-terminal amino acids (Pre46AA) that does not exist in the mature PtCA. In the present study, the function of the Pre46AA for the intracellular sorting of PtCA and the location of PtCA were determined. The sequence of the pre138 was ligated with egfp, which encodes the enhanced green fluorescent protein, packaged in the vector prasmid, Ppha-T1, downstream of promoter region of fucoxanthin chlorophylla/c binding protein gene (PfcpA), and introduced into P. tricornutum by microprojectile bombardment. Subsequently expressed Pre46AA-GFP fugion protein was shown to be localized in the whole chloroplast whereas GFP without Pre46AA was localized in cytoplasm. The function of the mature region of PtCA for the localization of PtCA in the chloroplast was further determined. pre138 was replaced with full length ptca cDNA in the constract described above and expressed in P. triconutum. The chimeric protein of PtCA-GFP was shown to be localized in storoma as 2 to 6 particles per chloroplast. These particles were sized at about 1 m and were localized aside thylakoid membranes. Histochemical staining of mitochondria with Mito-Tracker^TM clearly showed that these particles are different from mitochondria. These results indicate that The Pre46AA is required for the sorting of PtCA into chloroplast through chloroplast ER membranes and chloroplast envelopes and that the mature region of PtCA seems to function to stick together to form storomal particles.

KEY WORDS: carbonic anhydrase, pyrenoid, marine diatom, protein sorting