An optogenetic system to control membrane phospholipid asymmetry through flippase activation in budding yeast

Lipid asymmetry in biological membranes is essential for various cell functions, such as cell polarity, cytokinesis, and apoptosis. P4-ATPases (flippases) are involved in the generation of such asymmetry. In Saccharomyces cerevisiae , the protein kinases Fpk1p/Fpk2p activate the P4-ATPases Dnf1p/Dnf2p by phosphorylation. Previously, we have shown that a blue-light-dependent protein kinase, phototropin from Chlamydomonas reinhardtii ( Cr PHOT), complements defects in an fpk1 Δ fpk2 Δ mutant. Herein, we investigated whether Cr PHOT optically regulates P4-ATPase activity. First, we demonstrated that the translocation of NBD-labelled phospholipids to the cytoplasmic leaflet via P4-ATPases was promoted by blue-light irradiation in fpk1 Δ fpk2 Δ cells with Cr PHOT. In addition, blue light completely suppressed the defects in membrane functions (such as endocytic recycling, actin depolarization, and apical-isotropic growth switching) caused by fpk1 Δ fpk2 Δ mutations. All responses required the kinase activity of Cr PHOT. Hence, these results indicate the utility of Cr PHOT as a powerful and first tool for optogenetic manipulation of P4-ATPase activity.