title: Phosphatidylserine stimulation of Drs2p·Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol-4-phosphate.
abstract: Here, Drs2p, a yeast lipid translocase that belongs to the family of P(4)-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-(32)P]ATP of a (32)P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here.
authors.affiliation: UMR 8221 (Systèmes Membranaires, Photobiologie, Stress et Détoxication), CNRS, Gif-sur-Yvette, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: Jacquot
authors.forename: Cédric
authors.initials: C
authors.lastname: Montigny
authors.forename: Hanka
authors.initials: H
authors.lastname: Hennrich
authors.forename: Raphaëlle
authors.initials: R
authors.lastname: Barry
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.forename: Christine
authors.initials: C
authors.lastname: Jaxel
authors.forename: Joost
authors.initials: J
authors.lastname: Holthuis
authors.forename: Philippe
authors.initials: P
authors.lastname: Champeil
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lenoir
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014638:Vanadates / Q000494:pharmacology
chemical_list: C427246:CDC50 protein, S cerevisiae; C104014:DRS2 protein, S cerevisiae; D003902:Detergents; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D010761:Phosphorus Radioisotopes; D029701:Saccharomyces cerevisiae Proteins; C037178:phosphatidylinositol 4-phosphate; D001224:Aspartic Acid; D014638:Vanadates; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D005459:Fluorides