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Vol. 19, Issue 5, 2069-2082, May 2008

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Yeast ARV1 Is Required for Efficient Delivery of an Early GPI Intermediate to the First Mannosyltransferase during GPI Assembly and Controls Lipid Flow from the Endoplasmic Reticulum

Kentaro Kajiwara^*, Reika Watanabe, Harald Pichler^,, Kensuke Ihara^*, Suguru Murakami^*, Howard Riezman, and Kouichi Funato^*

*Department of Bioresource Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; Department of Biochemistry, University of Geneva, Sciences II, CH-1211 Geneva, Switzerland; and Institute of Molecular Biotechnology, Graz University of Technology, A-8010 Graz, Austria

Submitted August 2, 2007; Revised February 1, 2008; Accepted February 8, 2008
Monitoring Editor: Sean Munro

Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.

Address correspondence to: Kouichi Funato (kfunato{at}hiroshima-u.ac.jp)

Abbreviations used: AbA, aureobasidin A; CFW, calcofluor white; CP, complete GPI precursor; CPY, carboxypeptidase Y; DHS, dihydrosphingosine; Dol-P-Man, dolicholphosphomannose; EtNP, phosphorylethanolamine; GDP-Man, GDP-mannose; GlcNAc-PI, N-acetylglucosaminyl-phosphatidylinositol; GlcN-PI, glucosaminyl-phosphatidylinositol; GlcN-acylPI, glucosaminyl-acyl-phosphatidylinositol; GPI, glycosylphosphatidylinositol; GPI-MT, GPI mannosyltransferase; HA, hemagglutinin; IPC, inositolphosphorylceramide; LY, lucifer yellow; MIPC, mannosyl inositolphosphorylceramide; M(IP)₂C, mannosyl di(inositolphosphoryl)ceramide; PI, phosphatidylinositol; PLC, phospholipase C; PLD, phospholipase D; UDP-GlcNAc, UDP-N-acetylglucosamine.