Protein kinases Fpk1p and Fpk2p are novel regulators of phospholipid asymmetry.
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 cdc50Delta 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 lem3Delta/dnf1Delta dnf2Delta 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 fpk1Delta fpk2Delta 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.
Nakano
Kenzi
K
Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate Schools of Medicine, Sapporo 060-0815, Japan.
Yamamoto
Takaharu
T
Kishimoto
Takuma
T
Noji
Takehiro
T
Tanaka
Kazuma
K
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D011992:Endosomes / Q000378:metabolism; D005800:Genes, Fungal; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021601:trans-Golgi Network / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C427246:CDC50 protein, S cerevisiae; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C533049:Fpk1 protein, S cerevisiae; C533050:Fpk2 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C480602:Dnf2 protein, S cerevisiae; C480601:Dnf1 protein, S cerevisiae