Type IV P-type ATPases distinguish mono- versus diacyl phosphatidylserine using a cytofacial exit gate in the membrane domain

Abstract

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.

FIGURE 1.

Dnf1 variants differ in their suppression of drs2Δ PS asymmetry defects and cold-sensitive growth. A–C, a drs2Δ strain harboring either an empty vector or a vector expressing the indicated Drs2 or Dnf1 variant, were grown for 20 h at 30 °C with increasing concentrations of papuamide B (A and C) or duramycin (B). Among Dnf1 variants competent for NBD-PS transport, Dnf1 N550S is uniquely capable of restoring plasma membrane asymmetry in drs2Δ better than WT Dnf1 for PS asymmetry (A) but not PE asymmetry (B). C, overexpressing WT Dnf1 or Dnf1[Y618F] provides minor suppression of the PS asymmetry defect. D, growth of drs2Δ harboring the indicated Drs2 or Dnf1 variants at 30 °C or 20 °C. Dnf1 N550S suppresses drs2Δ cold-sensitive growth better than WT Dnf1 or other Dnf1 NBD-PS-transporting alleles.

FIGURE 2.

Dnf1 suppressors of drs2Δ cold-sensitive growth transport NBD-PS across the plasma membrane and restore endogenous PS asymmetry. A, growth of drs2Δ expressing the indicated Drs2 and Dnf1 variants at 30 °C or 20 °C. B, dnf1Δdnf2Δ cells expressing the indicated Dnf1 variants were incubated with NBD-phospholipid for 30 min at 4 °C as described under “Experimental Procedures.” The amount of lipid transported was normalized to NBD-PC uptake by WT Dnf1. C, the data shown in B was graphed as the ratio of NBD-PE to NBD-PC uptake (open bars) or NBD-PS to NBD-PC uptake (gray bars) to assess relative changes in substrate preference. The dotted lines are the ratios for WT Dnf1, and the shaded region above and below these lines are confidence intervals. Values extending outside of the confidence intervals represent a significant change in substrate specificity. D, the Dnf1 variants indicated were tested for their ability to restore PapB resistance (PS asymmetry) to a drs2Δ strain. The Dnf1 suppressors of drs2Δ cold-sensitive growth restore PS asymmetry better than WT Dnf1.

FIGURE 3.

Dnf1 suppressors of drs2Δ flip diacyl PS. A, localization of the PS-specific probe GFP-LactC2 in the WT strain or PS-deficient strain (cho1Δ). In WT yeast, PS recruits GFP-LactC2 to the plasma membrane, whereas in the cho1Δ strain, GFP-LactC2 is present primarily in the cytosol. The plot of pixel intensity across the lines in the images indicates that GFP-Lac2 is present at the plasma membrane in WT cells but not cho1Δ. B, a cho1Δdrs2Δ PS-deficient strain expressing GFP-LactC2 and the indicated Dnf1 variants were incubated with 10 μm monoacyl lyso-PS or 100 μm DLPS at 4 °C for the times indicated. The ability of each strain to translocate unlabeled PS substrate across the membrane is indicated by recruitment of GFP-LactC2 from the cytosol to the inner leaflet of the plasma membrane. Dnf1 N550S and Dnf1 T254A can flip dually acylated PS (DLPS) to the cytosolic leaflet, but WT Dnf1 and Dnf1[Y618F] cannot. The right column is a plot of pixel intensity along the lines drawn across cell buds at 150 min. Scale bars = 10 μm.

FIGURE 4.

Requirement for Dnf1, Dnf2, and Drs2 in the translocation of lyso-PS and DLPS. A, a cho1Δdnf1Δdnf2Δ strain expressing GFP-LactC2 was incubated with 10 μm lyso-PS at 4 °C for the times indicated prior to imaging. Transport of lyso-PS was significantly slower than with the strains used for Fig. 3 that expressed Dnf1 and Dnf2. B, at 30 °C, a condition permitting endocytosis, both lyso-PS and DLPS are flipped to the cytosolic leaflet of a cho1Δdnf1,2Δ strain. C, a cho1Δdrs2Δ strain carrying an empty plasmid or expressing Drs2[QQ→GA] failed to transport DLPS to the cytosolic leaflet at 30 °C. Drs2[QQ→GA] is specifically deficient for PS recognition. Scale bars = 10 μm.

FIGURE 5.

Substitutions of specific TM1–3 residues restore function to a Dnf1 P + 4 leucine variant. A, sequence alignment of TM1–2 and TM3–4 from lyso-PC flippases (Dnf1 and Dnf2), PS flippases (Drs2, Atp8a1, Atp8a2), and Ca²⁺ ATPase. The blue boxes include residues reported previously as contributing to PS specificity. The red boxes indicate newly identified residues contributing to PS specificity in Dnf1 mutants that correspond to equivalent Atp8a1 residues. B, double mutants combining a p + 4 leucine (Y618L) with Atp8a1 residues (F213S, T254A, or D258E) allows Dnf1 complementation of a dnf1,2,3Δdrs2Δ strain. The 5-fluoroorotic acid kills cells that were unable to lose the URA3-marked plasmid carrying DRS2 in the parental strain. Strains that grow express a Dnf1 variant that can support the viability of dnf1,2,3Δdrs2Δ in the absence of Drs2. C, the same set of Dnf1 variants were tested for the ability to support growth of drs2Δ at 20 °C. D, similarly, each Y618L double mutant tested partially restored membrane PS asymmetry to drs2Δ relative to Y618L alone.

FIGURE 6.

Drs2 functions are restored with a P + 4 leucine (F511L) when combined with substitutions corresponding to Atp8a1 residues. A, in Drs2, substitutions of Atp8a1 residues combined with F511L partially restores the ability to complement a dnf1,2,3Δdrs2Δ strain. The same Drs2 variants restore cold resistance to drs2Δ (B) and support membrane PS asymmetry (E) better than Drs2 F511L alone. Substituting the individual residues also allows Drs2 to support growth of dnf1,2,3Δdrs2Δ (C) and cold-resistant growth of drs2Δ (D). 5-FOA, 5-fluoroorotic acid.

FIGURE 7.

Cooperation between the P + 4 position in TM4 and residues in TM1–3 for PS selection. A, the Dnf1 residues highlighted in purple can be mutated to residues that allow PS transport. Mutations in the highlighted TM1 and 2 positions to Atp8a1 residues can rescue function of a Drs2 or Dnf1 P + 4 leucine mutant. B–D, Ca²⁺ ATPase crystal structure in the E2 conformation with a PE molecule cocrystallized. The equivalent residues contributing to phospholipid specificity in Dnf1 and Drs2 have been color-coded for reference: cyan, Asp-254 (equivalent to Dnf1 Asn-550, Drs2 Asn-445); red, Pro-312 (Dnf1 Tyr-618, Drs2 Phe-511); pink, Ile-315 (Dnf1 Val-621, Drs2 Val-514); orange, Thr-316 (Dnf1 Glu-622, Drs2 Glu-515); blue, Asn-101 (Dnf1 Thr-254, Drs2 Ser-261); and yellow, Gly-105 (Dnf1 Asp-258, Drs2 Glu-265) (PDB code 2AGV (46).