Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Abstract

Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the "giant substrate problem" because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.

Fig. 1.

Transmembrane segments 3–4 from Drs2 are sufficient to confer a PS activity to Dnf1. (A) Topology diagram of a P4-ATPase; A, actuator domain; N, nucleotide-binding domain; P, phosphorylation domain; LL3-4, lumenal loop between TM3 and TM4; black lines denote the membrane boundaries. (B) NBD-PL uptake across the plasma membrane mediated by Dnf1[Drs2] chimeras. TM3-4 contains important sequence for phospholipid recognition. Results represent averages of at least three independently isolated transformants from at least three independent experiments (mean ± SEM). (C) Localization of N-terminal GFP fused Dnf1 and Dnf1[Drs2] chimeras in S. cerevisiae. GFP-Dnf1 is localized to intracellular punctae in nonbudded cells (a, d), to the bud tip in small-budded cells (a, c) and to the bud neck in large-budded cells (b, d). A subset of GFP Dnf1[Drs2] chimeras mislocalize to the ER (e–g). Arrowheads designate polarized expression patterns (bud tip or bud neck); open arrowheads show punctate localization; arrows indicate perinuclear ER fluorescence. Fluorescence intensities for the images are scaled independently to emphasize localization. (Scale bar: 10 μm.)

Fig. 2.

Drs2 TM3-4 confers PS preference to Dnf1, while TM4 confers a PS activity without affecting the recognition of PC or PE. (A) NBD-PL uptake shows TM4 from Drs2 is sufficient to confer a PS activity to Dnf1, while TM3 and LL3-4 are important for NBD-PC and NBD-PE selection (mean ± SEM). (B) The ratio of PS to PC uptake from (A) was plotted to provide a measure of substrate preference that is independent of expression level. (C) Localization of GFP tagged Dnf1[TM3], Dnf1[LL3-4], and Dnf1[TM4] chimeras in S. cerevisiae indicate these chimeras localize and traffic similarly to WT Dnf1. (Scale bar: 10 μm.)

Fig. 3.

A single amino acid change, Y618F, specifically confers PS uptake activity to Dnf1. (A) Primary sequence alignment of TM4 from Dnf1, Dnf2, Drs2, and Atp8a1. Underlined sequences are the predicted TM4; boxes are regions differing between Dnf1 and Drs2. (B) NBD-PL uptake by Dnf1[Drs2] chimeras. Dnf1[YIS → FVT] and Dnf1 Y618F both transport PS (mean ± SEM). (C) PS/PC uptake ratio representing the relative preference of each chimera for either PC or PS independent of expression. (D) NBD-lipid uptake suggests Dnf1[TM3-4], Dnf1[TM4], and Dnf1 Y618F retain specificity for the phospholipid headgroup and glycerol backbone. Dnf1[YIS → FVT] is less selective than other PS transporting chimeras based on its activity for NBD-SM. Each reported value (B–D) is the average of at least three independent samples from at least three independent experiments (mean ± SEM).

Fig. 4.

Drs2 F511Y specifically perturbs plasma membrane PS asymmetry. (A) Growth of a drs2Δ strain expressing an empty vector, WT Drs2, Drs2 F511Y, or Drs2 F511L in the presence of papuamide B demonstrates a partial loss of PS asymmetry with Drs2 F511Y and Drs2 F511L (mean ± SEM). (B) Growth of a drs2Δ strain expressing the same constructs in the presence of duramycin indicates plasma membrane PE asymmetry is maintained by WT Drs2 and Drs2 F511Y but is perturbed with Drs2 F511L (mean ± SEM). (C) Drs2 F511Y retains an ATPase specific activity comparable to WT Drs2 while Drs2 F511L has a reduced ATPase activity (mean ± SD). (D) Drs2 F511Y fully complements cold-sensitive growth defect of a drs2Δ strain but Drs2 F511L only partially complements.

Fig. 5.

The rate of NBD-PC and NBD-PS uptake mediated by Dnf1 Y618F is comparable, suggesting a similar transport mechanism. (A) NBD-PC uptake at 0, 10, 30, and 60 min where 100% corresponds to WT Dnf1 NBD-PC uptake at 1 h (mean ± SEM). (B) NBD-PS uptake where 100% corresponds to WT Dnf1 NBD-PC uptake at 1 h (mean ± SEM).

Fig. 6.

F587 substitutions specifically perturb PC uptake activity by Dnf1. (A) Primary sequence alignment of TM3-LL3-4 from Dnf1, Dnf2, Drs2, and Atp8a1. Underlined sequences are the predicted TM3 and TM4; box indicate F587 in Dnf1. (B) Growth of dnf1,2,3Δdrs2Δ expressing the Dnf1 constructs indicated on synthetic defined (SD) media, SD plus edelfosine, SD plus 5FOA. (C) NBD-PL uptake indicates a defect in PC uptake by Dnf1 F587Y and Dnf1 F587L (p < 0.05) and no defect in PE uptake (p > 0.20) (mean ± SEM).

Fig. 7.

GFP tagged Dnf1[Drs2] chimeras retain a Lem3 requirement for ER export and flippase activity. (A) GFP tagged chimeras were expressed in lem3Δ cells (SCY119) overexpressing CDC50 or LEM3. Each Dnf1[Drs2] chimera tested exhibited a characteristic ER localization pattern in lem3∆ cells overexpressing CDC50. In contrast, overexpression of LEM3 in a lem3Δ strain rescues each Dnf1[Drs2] chimera polarized localization pattern. In each row, the fluorescence intensity of each image is scaled equivalently to show relative expression levels of each chimera. (Scale bar: 10 μm.) (B) NBD-PL uptake by Dnf1[Drs2] chimeras expressed in dnf1,2Δcdc50Δ or dnf1,2Δlem3Δ strains.

Fig. 8.

Model of the Dnf1 structure showing the orientation of Y618 and a proposed pathway for phospholipid translocation. (A) Na⁺/K⁺ ATPase crystal structure in the E2(2K⁺)-P_i conformation [PDB 2ZXE (3)]. Dark gray lines represent the membrane boundaries. (B) View of the Na⁺/K⁺ ATPase TM domain from the cytosolic side. (C) Structural model of Dnf1 highlighting segments involved in phospholipid selection and the groove between TM1, TM3, and TM4 forming a potential pathway for phospholipid flip. (D) View of the Dnf1 TM domain from cytosolic side of bilayer. Tyr618 is oriented on the opposite side of TM4 relative to the cation binding pocket of ion-transporting P-type ATPases. (E) Dnf1 model rotated 90° compared to (C). The yellow arrow represents the potential phospholipid transport pathway at the protein/lipid interface between TM1,TM3, and TM4. (F) Triangular cleft formed from TM1, TM3, LL3-4, and TM4. Arrows indicate F587 and Y618 of Dnf1, residues involved in PC and PS selection, respectively.

Fig. P1.

Regions of the flippase Dnf1 called transmembrane segments 3–4 (TM3-4) are responsible for phospholipid selection. We propose that the phospholipid headgroup is selected and transported along a groove between TM1, 3, and 4, allowing the hydrophobic lipid tail to simply reorient in the hydrophobic core of the membrane bilayer. TM3-4 is highlighted in purple within this homology model of the Dnf1 structure.