title: Regulation of a Golgi flippase by phosphoinositides and an ArfGEF.
abstract: 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. 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, 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. 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.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
authors.forename: Paramasivam
authors.initials: P
authors.lastname: Natarajan
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.forename: Dustin V
authors.initials: DV
authors.lastname: Patil
authors.forename: Vicki A
authors.initials: VA
authors.lastname: Sciorra
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Jackson
authors.forename: Todd R
authors.initials: TR
authors.lastname: Graham
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: C104014:DRS2 protein, S cerevisiae; C121632:GEA2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D010716:Phosphatidylinositols; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases