Purification, characterization and reconstitution of Drs2 protein, a phospholipid flippase from budding yeast

Abstract

Type-IV P-type ATPases are putative phospholipid flippases that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. However, flippase activity has not been reconstituted with any purified type-IV P-type ATPase, and so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. The major goal of this dissertation project is to test whether Drs2p, a type-IV P-type ATPase from yeast Saccharomyces cerevisiae, is a flippase or not. I show that Drs2p can directly catalyze phospholipid translocation through purification and reconstitution of this ATPase into artificial membranes. This flippase activity is specific to a phosphatidylserine analogue and requires active Drs2p. My data also suggest that only a portion of Drs2p molecules are active after purification and these active molecules may result from partial proteolysis within the carboxyl terminus of Drs2p. This observation is consistent with a model that the carboxyl tail is auto-inhibitory to Drs2p activity. In this study, I demonstrate for the first time the reconstitution of flippase activity with a purified type-IV P-type ATPase.