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

Zhou X, Graham TR
Proc Natl Acad Sci U S A. 2009 106 (39): 16586-91

PMID: 19805341 · PMCID: PMC2757829 · DOI:10.1073/pnas.0904293106

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.

MeSH Terms (6)

Adenosine Triphosphatases Calcium-Transporting ATPases Phospholipids Phospholipid Transfer Proteins Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins

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