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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.

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.