title: Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity.
abstract: Lipid asymmetry at the plasma membrane is essential for such processes as cell polarity, cytokinesis and phagocytosis. Here we find that a lipid flippase complex, composed of Lem3, Dnf1 or Dnf2, has a role in the dynamic recycling of the Cdc42 GTPase, a key regulator of cell polarity, in yeast. By using quantitative microscopy methods, we show that the flippase complex is required for fast dissociation of Cdc42 from the polar cortex by the guanine nucleotide dissociation inhibitor. A loss of flippase activity, or pharmacological blockage of the inward flipping of phosphatidylethanolamine, a phospholipid with a neutral head group, disrupts Cdc42 polarity maintained by guanine nucleotide dissociation inhibitor-mediated recycling. Phosphatidylethanolamine flipping may reduce the charge interaction between a Cdc42 carboxy-terminal cationic region with the plasma membrane inner leaflet, enriched for the negatively charged lipid phosphatidylserine. Using a reconstituted system with supported lipid bilayers, we show that the relative composition of phosphatidylethanolamine versus phosphatidylserine directly modulates Cdc42 extraction from the membrane by guanine nucleotide dissociation inhibitor.
authors.affiliation: Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
authors.forename: Arupratan
authors.initials: A
authors.lastname: Das
authors.forename: Brian D
authors.initials: BD
authors.lastname: Slaughter
authors.forename: Jay R
authors.initials: JR
authors.lastname: Unruh
authors.forename: William D
authors.initials: WD
authors.lastname: Bradford
authors.forename: Richard
authors.initials: R
authors.lastname: Alexander
authors.forename: Boris
authors.initials: B
authors.lastname: Rubinstein
authors.forename: Rong
authors.initials: R
authors.lastname: Li
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D020730:Guanine Nucleotide Dissociation Inhibitors; C478771:Lem3 protein, S cerevisiae; D008563:Membrane Lipids; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; C534963:Rdi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C483858:phosphatidylethanolamine; D000251:Adenosine Triphosphatases; C480602:Dnf2 protein, S cerevisiae; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; C480601:Dnf1 protein, S cerevisiae