P4 ATPases: flippases in health and disease

Abstract

P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed "lipid flipping". Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.

Figure 1

Phylogenetic tree of the P-type ATPase superfamily and of the P4 branch. Substrates of P1–P5 branches are in between brackets. Phylogenetic analyses of the P4 ATPase protein family of mammalian, A. thaliana, S. cerevisiae, and C. elegans is shown and was compiled using ClustalW sequence alignment software [43]. Database accession numbers: C. elegans: TAT-1 (NP_001022894), TAT-2 (NP_001023252), TAT-3 (NP_499363), TAT-4 (NP_495244), TAT-5 (NP_001021457), TAT-6 (NP_503858); A. thaliana: ALA1 (P98204), ALA2 (P98205), ALA3 (Q9XIE6), ALA4 (Q9LNQ4), ALA5 (Q9SGG3), ALA6 (Q9SLK6), ALA7 (Q9LVK9), ALA8 (Q9LK90), ALA9 (Q9SX33), ALA10 (Q9LI83), ALA11 (Q9SAF5), ALA12 (P57792); S. cerevisiae: Drs2p (P39524), Dnf1p (P32660), Dnf2p (Q12675), Dnf3p (Q12674), Neo1p (P40527). H. sapiens: ATP8A1 (P70704), ATP8A2 (P98200), ATP8B1 (O43520), ATP8B2 (P98198), ATP8B3 (O60423), ATP8B4 (Q8TF62), ATP9A (O75110), ATP9B (O43861), ATP10A (O60312), ATP10B (O94823), ATP10D (Q9P241), ATP11A (P98196), ATP11B (Q9Y2G3), ATP11C (Q8NB49).

Figure 2

Simplified topological model of a P4 ATPase and its CDC50 subunit. P4 ATPases consist of an actuator (A), phosphorylation (P), nucleotide binding (N) and 10 predicted membrane spanning helices. CDC50 subunits consist of 2 membrane spanning domains with a large extracellular loop containing four possible N-linked glycosylation sites and two disulfide bridges. Modified from Coleman et al.[44].

Figure 3

Proposed reaction cycles of a P4 ATPase (a) and a P2C ATPase (Na⁺/K⁺ ATPase) (b) complexed with their subunit. P-type ATPases cycle through four main separate conformations when transporting ligands. In the E1 state the P-type ATPases have high affinity for intracellular ligands; Na⁺ in the case of the Na⁺/K⁺ ATPase, unknown or none for the P4 ATPase. Binding of ATP to the N-domain and subsequent phosphorylation of the P domain results in the E1-P state. While converting from E1-P to E2-P, intracellular ligands (3 Na⁺ for the Na⁺/K⁺ ATPase) are released into the exoplasmic milieu and the A-domain rotates. This allows binding of extracellular ligands (2 K⁺ for the Na⁺/K⁺ ATPase) or a phospholipid (depicted in pink) from the exoplasmic leaflet. Affinity for the subunit is highest in this state and this interaction may assist in binding of the phospholipid. Dephosphorylation changes the enzyme from the E2-P to the E2 state. Movement of the A-domain away from the P-domain reverts the ATPase back to the E1 state thereby translocating the extracellular ligands or the phospholipid to the cytoplasmic side. Adapted from Coleman et al. and Lenoir et al.[44,65].

Figure 4

Proposed roles of P4 ATPases in intracellular vesicle trafficking routes in S. cerevisiae, A. thaliana and C. elegans. Although many P4 ATPases are linked to intracellular trafficking defects, only a few have been specifically linked to certain organelles. S. cerevisiae Neo1p has been implicated in retrograde, COPI-dependent trafficking from the Golgi to the ER [99,100]. Drs2p in yeast is involved in the formation of AP-1/clathrin coated vesicles back and forth between the TGN and early endosomes [40,54,62,86,87]. Yeast Dnf1p and Dnf2p play a role in the formation of endocytic vesicles [62,96,97]. A. thaliana ALA3 is necessary for the synthesis of secretory vesicles from the TGN [39]. C. elegans TAT-1 is important in maintaining normal endocytic recycling and biogenesis of lysosomes [32,102] whereas TAT-5 was suggested to be involved in the regulation of ectosome shedding [103]. In Chinese hamster ovary cells ATP8A1 plays a role in cell migration by assisting in the formation of plasma membrane ruffles [104]. ATP8B1 is necessary for apical hepatocyte membrane integrity in M. musculus[108,109,126]. Possible functional locations of P4-ATPases are represented by colored circles; red for S. cerevisiae, yellow for A. thaliana, green for C. elegans and blue for mammalian P4-ATPases. See text for further details.