Two-gate mechanism for phospholipid selection and transport by type IV P-type ATPases

Proc Natl Acad Sci U S A. 2013 Jan 29;110(5):E358-67. doi: 10.1073/pnas.1216948110. Epub 2013 Jan 9.


Most P-type ATPases pump specific cations or heavy metals across a membrane to form ion gradients. However, the type IV P-type ATPases evolved the ability to transport specific phospholipid substrates rather than cations and function to establish plasma membrane asymmetry in eukaryotic cells. The mechanism for how a P-type ATPase, or any other transporter, can recognize and flip a phospholipid substrate is unclear. Here, through a combination of genetic screening and directed mutagenesis with the type IV P-type ATPases Dnf1 and Drs2 from budding yeast, we identify more than a dozen residues that determine headgroup specificity for phospholipid transport. These residues cluster at two interfacial regions flanking transmembrane segments 1-4 and lie outside of the canonical substrate binding site operating in cation pumps. Our data imply the presence of two substrate-selecting gates acting sequentially on opposite sides of the membrane: an entry gate, where phospholipid is initially selected from the extracellular leaflet, and an exit gate at the cytosolic leaflet. The entry and exit gates act cooperatively but imperfectly, with neither being able to restrict phosphatidylserine selection completely when the opposing gate is tuned to permit it. This work describes a unique transport mechanism for a P-type ATPase and provides insight into how integral membrane proteins can recognize and transport phospholipid substrate across a lipid bilayer.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism*
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Amino Acids / genetics
  • Amino Acids / metabolism
  • Binding Sites / genetics
  • Biological Transport / drug effects
  • Biological Transport / genetics
  • Blotting, Western
  • Calcium-Transporting ATPases / chemistry
  • Calcium-Transporting ATPases / genetics
  • Calcium-Transporting ATPases / metabolism*
  • Cell Membrane / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Lipid Bilayers / metabolism
  • Microscopy, Fluorescence
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Phosphatidylcholines / chemistry
  • Phosphatidylcholines / metabolism
  • Phosphatidylserines / chemistry
  • Phosphatidylserines / metabolism
  • Phospholipid Ethers / pharmacology
  • Phospholipids / chemistry
  • Phospholipids / metabolism*
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Substrate Specificity


  • ATP-Binding Cassette Transporters
  • Amino Acids
  • DRS2 protein, S cerevisiae
  • Lipid Bilayers
  • Phosphatidylcholines
  • Phosphatidylserines
  • Phospholipid Ethers
  • Phospholipids
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • edelfosine
  • Adenosine Triphosphatases
  • Calcium-Transporting ATPases
  • Dnf1 protein, S cerevisiae