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.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…Some PIP-binding proteins help regulate the secretory pathway [46,59]. A type IV P-type ATPase, required for flippase activity, is a PI(4)P effector [62], suggesting that increased PI(4)P levels (Fig. 10a) may enhance flippase activity resulting in an unfavorable local lipid environment to support fusion.…”

A P4-ATPase Protein Interaction Network Reveals a Link between Aminophospholipid Transport and Phosphoinositide Metabolism

“…Proteins captured on streptavidin beads were subjected to on-bead Lys-C/trypsin digestion essentially as described in Guerrero et al 9 Lys-C/trypsin digests were desalted using Stage-tips (50), and analyzed on a nanoflow LC-MS/MS system using a LTQ ion trap mass spectrometer (Thermo Electron, Bremen) operated as described. 51 All MS/MS spectra were converted to single DTA files using Bioworks 3.1 (Thermo) with default parameters, and merged into a file in Mascot generic format. This was searched against the yeast SGD database (5779 entries, obtained from http://www.yeastgenome.org, June 2007) using a Mascot search engine (version 2.2.03).…”

Arl1p regulates spatial membrane organization at the trans -Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p

“…Drs2p is an aminophospholipid translocase that facilitates the translocation of phosphatidylserine (PS) from the luminal to the cytosolic leaflet of the membrane at the TGN (21,33). Thus, we first examined whether the flippase activity of Drs2p is required for the Golgi localization of Imh1p.…”