P4-ATPases: lipid flippases in cell membranes

Abstract

Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids and synthetic alkylphospholipids. At the same time, the cellular processes known to be directly or indirectly affected by this class of transporters have expanded to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell.

Fig. 1

Membrane topology of P2- and P4-ATPases and their subunits. P-type ATPases consist of an actuator (A), a phosphorylation (P), a nucleotide-binding domain (N), and 10 transmembrane spanning helices. The P domain contains the canonical aspartic acid phosphorylated during the reaction cycle. The beta subunits associated with P2-ATPases are type II membrane proteins with one transmembrane segment, a short cytoplasmic tail, and a large, heavily glycosylated ectodomain with three disulfide bridges. In some cases, a gamma subunit belonging to the FXYD protein family is associated to the P2-ATPases. The CDC50 subunits of P4-ATPases consist of two membrane-spanning domains with a large extracellular loop containing four possible N-linked glycosylation sites and two disulfide bridges. Both the membrane and extracellular domains of CDC50 are required for assembly with the P4-ATPase [17, 68]

Fig. 2

Cellular functions involving P4-ATPases. P4-ATPases appear to exert their cellular functions by combining an enzymatic phospholipid translocation activity with an enzyme-independent action. These functions are not mutually exclusive. Active transport of lipids from the exoplasmic to the cytosolic membrane leaflet can maintain lipid asymmetry (a), scavenge lipids (b), and drive membrane budding by generating a lipid imbalance across the bilayer and/or a membrane environment permissive for vesicle budding (c). Enzyme-independent functions of P4-ATPases include recruitment of proteins involved in coat assembly (d), cellular signaling, and cytoskeleton regulation (e). See text for details

Fig. 3

Schematic overview of two proposed phospholipid transport pathways in P4-ATPases. In the classical model, the lipid is transported through a space in the transporter analogous to the cation transport mechanism of well-characterized P2-ATPases. Here, an occluded state is expected with the transported lipid deeply buried in a central cavity (red) within the P4-ATPase with the entrance and exit pathways closed. By contrast, in the external surface model, the lipid is transported at a cleft on the membrane-facing surface, and only the lipid head group is protected from the lipid environment. The presence of two substrate-selecting gates (green) acting sequentially on opposite sides of the membrane has been reported [4]. In both cases, the relative positioning of the transmembrane segments critical for phospholipid binding/transport is highlighted on a homology model of Dnf1p based on the crystallized Na⁺/K⁺-ATPase in the E2P conformation [3]. The rest of the structure is shown in surface representation