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 (CrPHOT), complements defects in an fpk1Δ fpk2Δ mutant. Herein, we investigated whether CrPHOT 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 fpk1Δ fpk2Δ cells with CrPHOT. In addition, blue light completely suppressed the defects in membrane functions (such as endocytic recycling, actin depolarization, and apical-isotropic growth switching) caused by fpk1Δ fpk2Δ mutations. All responses required the kinase activity of CrPHOT. Hence, these results indicate the utility of CrPHOT as a powerful and first tool for optogenetic manipulation of P4-ATPase activity.