Regulation of a Golgi flippase by phosphoinositides and an ArfGEF

Abstract

SUMMARYThe essential role for phosphatidylinositol 4-phosphate (PtdIns[4]P) in vesicle-mediated protein transport from the trans-Golgi network (TGN) was first described in budding yeast1–3. However, the identity of downstream effectors of PtdIns[4]P in this system has been elusive. Here, we show that Drs2p, a type IV P-type ATPase required for phospholipid translocase (flippase) activity and transport vesicle budding from the TGN4–8, is an effector of PtdIns[4]P. Drs2p-dependent flip of a fluorescent phosphatidylserine analogue across purified TGN membranes requires synthesis of PtdIns[4]P by the PtdIns 4-kinase Pik1p. PtdIns[4]P binds to a regulatory domain in the C-terminal tail of Drs2p that has homology to a split PH domain and is required for Drs2p activity. In addition, basic residues required for phosphoinositide binding overlap a binding site for the ArfGEF Gea2p that was previously mapped9. ArfGEF binding to this C-terminal domain also stimulates flippase activity in TGN membrane preparations. These interactions imply the presence of a novel coincidence detection system used to activate phospholipid translocation at sites of vesicle formation.