title: 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.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.forename: Todd R
authors.initials: TR
authors.lastname: Graham
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C104014:DRS2 protein, S cerevisiae; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases