title: Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase.
abstract: Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235.
authors.forename: Bartholomew P
authors.identifier: 0000-0002-9033-9942
authors.initials: BP
authors.lastname: Roland
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235 tr.graham@vanderbilt.edu.
authors.forename: Todd R
authors.initials: TR
authors.lastname: Graham
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D019020:Directed Molecular Evolution; D000073659:Gain of Function Mutation; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013109:Sphingomyelins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D029701:Saccharomyces cerevisiae Proteins; D013109:Sphingomyelins; D001216:Asparagine; D000251:Adenosine Triphosphatases; C480601:Dnf1 protein, S cerevisiae
keywords: P4-ATPase; directed evolution; membrane asymmetry; sphingomyelin