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

Abstract

Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg 2؉ -ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.flippase ͉ membrane asymmetry ͉ P4-ATPase ͉ proteoliposome ͉ Cdc50p

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2

“…This enigma has been referred to as the "giant substrate problem" (13,14). On the other hand, it is now clear that no protein other than the P4-ATPase catalytic chain in association with its small CDC50 subunit (β-subunit) is required for the flippase function, because the flipping of specific phospholipids is retained after purification and reconstitution of these proteins in lipid vesicles (2,3,12). In Na + ,K + -ATPase and Ca 2+ -ATPase, the central helices, M4, M5, and M6, play major roles in cation binding Significance Membranes of eukaryotic cells retain an essential asymmetric distribution of phospholipids in the two leaflets, which depends on the translocation by P4-ATPases (flippases) of specific phospholipids across the lipid bilayer.…”

“…M embers of the P4 subfamily of P-type ATPases are known as flippases, because they transport (flip) specific phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane bilayer (1)(2)(3). Thus, they establish and maintain a physiologically essential asymmetry between the two leaflets, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) being concentrated in the cytoplasmic leaflet and phosphatidylcholine (PC) and sphingomyelin in the exoplasmic leaflet.…”

ATP9B, a P4-ATPase (a Putative Aminophospholipid Translocase), Localizes to the trans-Golgi Network in a CDC50 Protein-independent Manner

“…P4-ATPases are a subfamily of P-type ATPases and have been implicated in flipping aminophospholipids from the exoplasmic (lumenal) leaflet to the cytoplasmic leaflet (11)(12)(13)(14)(15). The yeast P4-ATPases (Drs2p, Neo1p, Dnf1p, Dnf2p, and Dnf3p) are all involved in protein transport in the secretory and endocytic pathways albeit at different stages (16).…”

Membrane protein reconstitution for functional and structural studies

“…Because of the high binding capacity, this technique can be used to remove almost all kinds of detergents. This technique has been successfully employed to reconstitute Ca 2+ -ATPase [28], Type-IV P-type ATPases [31], rat heart mitochondrial F1Fo ATP synthase [32], and Multidrug Transporter LmrP Protein [33] into liposomes.…”