Regulation of a Golgi flippase by phosphoinositides and an ArfGEF

The essential role for phosphatidylinositol-4-phosphate (PtdIns(4)P) in vesicle-mediated protein transport from the trans-Golgi network (TGN) was first described in the budding yeast Saccharomyces cerevisiae(1-3). 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 TGN(4-8), 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 phosphatidylinositol-4-kinase (PI(4)K) 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 previously mapped binding site for the ArfGEF Gea2p(9). ArfGEF binding to this C-terminal domain also stimulates flippase activity in TGN membrane preparations. These interactions suggest the presence of a coincidence detection system used to activate phospholipid translocation at sites of vesicle formation.