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Eukaryotic Cell, March 2009, p. 306-314, Vol. 8, No. 3
1535-9778/09/$08.00+0     doi:10.1128/EC.00257-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro{triangledown}

Régine Bosson, Isabelle Guillas,{dagger} Christine Vionnet, Carole Roubaty, and Andreas Conzelmann*

University of Fribourg, Department of Medicine, Ch. Du Musée 5, CH-1700 Fribourg, Switzerland

Received 30 July 2008/ Accepted 2 December 2008

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [3H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43{Delta} or mcd4{Delta} mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [3H]DHS into anchors in vitro. Moreover, gup1{Delta} microsomes incorporate [3H]DHS into the same abnormal anchor lipids as gup1{Delta} cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [3H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [3H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.

* Corresponding author. Mailing address: Division of Biochemistry, Chemin du Musée 5, CH-1700 Fribourg, Switzerland. Phone: 41 26 300 8630. Fax: 41 26 300 9735. E-mail: andreas.conzelmann{at}unifr.ch

{triangledown} Published ahead of print on 12 December 2008.

{dagger} Present address: Université de Paris 6, UPMC Univ. Paris 06, UMR 7180, PCMP, F-75005 Paris, France, and CNRS, UMR 7180, PCMP, F-75005 Paris, France.

Eukaryotic Cell, March 2009, p. 306-314, Vol. 8, No. 3
1535-9778/09/$08.00+0     doi:10.1128/EC.00257-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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