Presentation Information
[P04-564]The sorting mechanism of the pyrenoid luminal carbonic anhydrase
○Riho Nagamatsu1, Yusuke Matsuda1, Ginga Shimakawa2 (1. Grad. Sch. Sci. Tech., Kwansei Gakuin Univ. (Japan), 2. Grad. Sch. Sci., Univ. Osaka (Japan))
Keywords:
Diatom,PPT,CCM,CA,Photosynthesis
[Purpose]
The present study aimed to elucidate the mechanisms underlying Ptθ-CA1 targeting to the PPT lumen and PPT formation in the pennate marine diatom Phaeodactylum tricornutum.
[Method]
We generated a series of deletion constructs in which regions either upstream (N-terminus) or downstream (C-terminus) of the CA domain, denoted as the Cysteine–Glycine–Histidine-rich (CGHR). These constructs retain N-terminal chloroplast targeting signals and were fused to GFP at each C-terminus and expressed in the Ptθ-CA1 KO strain. Furthermore, the constructs were designed to preserve essential targeting signals while removing surrounding regions in a stepwise manner to test their contribution to localization. In addition, we analyzed the co-localization candidates of Ptθ-CA1 identified by immunoprecipitation, as well as candidate factors potentially involved in PPT formation identified through bioinformatic approaches by detections of ER-targeting signals, chloroplast-targeting signals, and thylakoid-targeting motifs. These candidates were selected to examine whether additional factors may contribute to PPT targeting or structural formation.
[Results]
The results clearly showed that neither N- nor C-terminal deletant proteins was not mislocated, indicating that the Ptθ-CA1 sequence does not contain the PPT sorting signal outside of the CGHR domain. One of co-precipitated proteins of Ptθ-CA1 localized throughout the chloroplast, while the other co-precipitated protein was localized to the chloroplast endoplasmic reticulum (CER), both showing weak evidence of direct interaction with Ptθ-CA1. Interestingly, a bioinformatically-selected protein was localized to the PPT.
[Consideration]
These results suggest that the N- and C-terminal regions of Ptθ-CA1 are not required for PPT targeting, and that other factors may mediate its localization. The chloroplast-wide localization of one candidate protein suggests its presence in both stroma thylakoids and PPT, potentially contributing to interaction with Ptθ-CA1. Furthermore, the PPT localization of the bioinformatically identified protein implies a possible role in PPT formation.
[Conclusion]
Our findings indicate that Ptθ-CA1 targeting to the PPT lumen is independent of its N- and C-terminal regions outside the CGHR domain, and suggest that interacting factors, particularly PPT-localized proteins, may play key roles in PPT targeting and formation mechanisms.
The present study aimed to elucidate the mechanisms underlying Ptθ-CA1 targeting to the PPT lumen and PPT formation in the pennate marine diatom Phaeodactylum tricornutum.
[Method]
We generated a series of deletion constructs in which regions either upstream (N-terminus) or downstream (C-terminus) of the CA domain, denoted as the Cysteine–Glycine–Histidine-rich (CGHR). These constructs retain N-terminal chloroplast targeting signals and were fused to GFP at each C-terminus and expressed in the Ptθ-CA1 KO strain. Furthermore, the constructs were designed to preserve essential targeting signals while removing surrounding regions in a stepwise manner to test their contribution to localization. In addition, we analyzed the co-localization candidates of Ptθ-CA1 identified by immunoprecipitation, as well as candidate factors potentially involved in PPT formation identified through bioinformatic approaches by detections of ER-targeting signals, chloroplast-targeting signals, and thylakoid-targeting motifs. These candidates were selected to examine whether additional factors may contribute to PPT targeting or structural formation.
[Results]
The results clearly showed that neither N- nor C-terminal deletant proteins was not mislocated, indicating that the Ptθ-CA1 sequence does not contain the PPT sorting signal outside of the CGHR domain. One of co-precipitated proteins of Ptθ-CA1 localized throughout the chloroplast, while the other co-precipitated protein was localized to the chloroplast endoplasmic reticulum (CER), both showing weak evidence of direct interaction with Ptθ-CA1. Interestingly, a bioinformatically-selected protein was localized to the PPT.
[Consideration]
These results suggest that the N- and C-terminal regions of Ptθ-CA1 are not required for PPT targeting, and that other factors may mediate its localization. The chloroplast-wide localization of one candidate protein suggests its presence in both stroma thylakoids and PPT, potentially contributing to interaction with Ptθ-CA1. Furthermore, the PPT localization of the bioinformatically identified protein implies a possible role in PPT formation.
[Conclusion]
Our findings indicate that Ptθ-CA1 targeting to the PPT lumen is independent of its N- and C-terminal regions outside the CGHR domain, and suggest that interacting factors, particularly PPT-localized proteins, may play key roles in PPT targeting and formation mechanisms.
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