Lipid Specific Activation of the Murine P₄-ATPase Atp8a1 (ATPase II)

Abstract

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.

Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast

“…3 D and A), consistent with the properties of purified mammalian Atp8a1 (16,17). The specific activity of TAP-Drs2p was 0.45 Ϯ 0.03 mol Pi released/min/mg Drs2p (V max , Fig.…”

“…The ATPase activity of TAP-Drs2p also was Mg 2ϩ -dependent (Fig. 3D), as reported for purified mammalian Atp8a1 (16,17).…”

“…However, this ATPase activity was insensitive to orthovanadate, a commonly used P-type ATPase inhibitor (16,17), and was completely inhibited by 1 mM azide (Fig. 1D).…”

“…A flippase activity toward spin-labeled PS and PE analogues has been reconstituted with a partially purified, but unidentified, Mg 2ϩ -ATPase from human red blood cells (14). ATPase II/Atp8a1, a Mg 2ϩ -ATPase that potentially catalyzes aminophospholipid translocase activity in bovine chromaffin granules (15), has been purified to homogeneity (16,17). Phylogenetic analysis of the ATPase II/Atp8a1 sequence indicates that it is a member of the P4-ATPase family, which includes Fic1/Atp8b1 and Drs2p from yeast (18,19).…”

The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

“…A subfamily of P-type ATPases (type 4 subfamily or aminophospholipid translocases) mediates the flip of glycerophospholipids. In mammals, ATP8A1, ATP8B1, and ATP8B3 are reportedly involved in PS translocation (Ujhazy et al, 2001;Wang et al, 2004;Paterson et al, 2006). In yeast, five proteins belong to this family, Dnf1 and Dnf2 in the plasma membrane and Neo1, Drs2, and Dnf3, which reside in the internal membranes (Pomorski et al, 2003).…”