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Vol. 19, Issue 4, 1783-1797, April 2008

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Protein Kinases Fpk1p and Fpk2p are Novel Regulators of Phospholipid Asymmetry

Kenzi Nakano^*, Takaharu Yamamoto, Takuma Kishimoto^*, Takehiro Noji^*, and Kazuma Tanaka

Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate Schools of *Medicine and Life Science, Sapporo 060-0815, Japan

Submitted July 9, 2007; Revised December 20, 2007; Accepted January 8, 2008
Monitoring Editor: Sandra Lemmon

Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50 mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3/dnf1 dnf2 mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1 fpk2 mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.

Address correspondence to: Kazuma Tanaka (k-tanaka{at}igm.hokudai.ac.jp).

Abbreviations used: DIC, differential interference contrast; GFP, green fluorescent protein; mRFP1, monomeric red fluorescent protein 1; 3HA, three tandem repeats of the influenza virus hemagglutinin epitope; GST, glutathione S-transferase; NBD, 7-nitrobenz-2-oxa-1,3-diazol-4-yl; NBD-PC, 1-palmitoyl-2-(6-NBD- aminocaproyl)-PC; NBD-PE, 1-palmitoyl-2-(6-NBD-aminocaproyl)-PE; NBD-PS, 1-palmitoyl-2-(6-NBD-aminocaproyl)-PS; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine; TGN, trans-Golgi network.