Type IV P-type ATPases (P4-ATPases) use the energy from ATP to "flip" phospholipid across a lipid bilayer, facilitating membrane trafficking events and maintaining the characteristic plasma membrane phospholipid asymmetry. Preferred translocation substrates for the budding yeast P4-ATPases Dnf1 and Dnf2 include lysophosphatidylcholine, lysophosphatidylethanolamine, derivatives of phosphatidylcholine and phosphatidylethanolamine containing a 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) group on the sn-2 C6 position, and were presumed to include phosphatidylcholine and phosphatidylethanolamine species with two intact acyl chains. We previously identified several mutations in Dnf1 transmembrane (TM) segments 1 through 4 that greatly enhance recognition and transport of NBD phosphatidylserine (NBD-PS). Here we show that most of these Dnf1 mutants cannot flip diacylated PS to the cytosolic leaflet to establish PS asymmetry. However, mutation of a highly conserved asparagine (Asn-550) in TM3 allowed Dnf1 to restore plasma membrane PS asymmetry in a strain deficient for the P4-ATPase Drs2, the primary PS flippase. Moreover, Dnf1 N550 mutants could replace the Drs2 requirement for growth at low temperature. A screen for additional Dnf1 mutants capable of replacing Drs2 function identified substitutions of TM1 and 2 residues, within a region called the exit gate, that permit recognition of dually acylated PS. These TM1, 2, and 3 residues coordinate with the "proline + 4" residue within TM4 to determine substrate preference at the exit gate. Moreover, residues from Atp8a1, a mammalian ortholog of Drs2, in these positions allow PS recognition by Dnf1. These studies indicate that Dnf1 poorly recognizes diacylated phospholipid and define key substitutions enabling recognition of endogenous PS.
Keywords: Flippase; Lipid Transport; Membrane Asymmetry; Membrane Function; Membrane Proteins; Membrane Transport; P4-ATPase; Phosphatidylserine; Yeast.