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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.

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  • Authors

    Jacquot A, Montigny C, Hennrich H, Barry R, le Maire M, Jaxel C, Holthuis J, Champeil P, Lenoir G

    Source

    The Journal of biological chemistry 287:16 2012 Apr 13 pg 13249-61

    MeSH

    Adenosine Triphosphate
    Aspartic Acid
    Calcium-Transporting ATPases
    Detergents
    Fluorides
    Phosphatidylinositol Phosphates
    Phosphatidylserines
    Phosphorus Radioisotopes
    Phosphorylation
    Plasmids
    Saccharomyces cerevisiae
    Saccharomyces cerevisiae Proteins
    Solubility
    Vanadates

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    22351780