1.
An optogenetic system to control membrane phospholipid asymmetry through flippase activation in budding yeast.
Abstract:
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.
2.
Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.
Abstract:
Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.
