Abstract
Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal, and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins; however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cδ1 and Akt1, but not Bruton’s tyrosine kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular localization of other proteins in P. yezoensis and other red algal cells.
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Acknowledgements
We are grateful to Dr. Tamas Balla (National Institute of Child Health and Human Development, National Institutes of Health, USA) for kindly providing the plasmids containing the PH domain from PLCδ1 and Btk. This study was supported in part by a grant from the Sumitomo Foundation (to K. M.) and by Grants-in-Aid for the 21st COE (Center Of Excellence) Program “Marine Bio-Manipulation Frontier for Food Production” (to N. S.) and City Area Program in Industry–Academia–Government Joint Research (Hakodate area) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to H. Y. and N. S.).
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Mikami, K., Uji, T., Li, L. et al. Visualization of Phosphoinositides via the Development of the Transient Expression System of a Cyan Fluorescent Protein in the Red Alga Porphyra yezoensis . Mar Biotechnol 11, 563–569 (2009). https://doi.org/10.1007/s10126-008-9172-z
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DOI: https://doi.org/10.1007/s10126-008-9172-z