Lipid
remodeling of glycosylphosphatidylinositol anchors in yeast Saccharomyces
cerevisiae.
Régine Bosson,
Isabelle Guillas, Andreas Conzelmann
Institute of Biochemistry, University of Fribourg, Ch. du Musée 5, Fribourg,
1700, Switzerland (regine.bosson@unifr.ch)
GPI-anchored
proteins of S. cerevisiae exist with two types of lipid moiety, either diacylglycerol
(DAG) or ceramide, both containing C26:0 fatty acids. Since the intermediates
of the GPI-anchor biosynthesis do not have these long chain fatty acids, the
lipids have to be exchanged. There are at least three different remodeling
pathways: (a) remodeling from DAG to ceramide in the ER; (b) remodeling from
DAG to a more hydrophobic DAG in the ER; and (c) remodeling to a more polar
ceramide in the Golgi (Sipos G. et al., EMBOJ 1997, 16(12): 3494-3505). The aim
of this project is to find the gene(s) encoding the remodelase(s). There are
different approaches leading to it: first, a selection for mutants that
mistarget GPIs into the vacuole, because we suppose that the GPIs whose lipids
have not been remodeled, would go to the vacuole; second, the lipid analysis of
the anchors of mutants that could be remodelase candidates; and third, an in
vitro assay to test the remodelase activity. Here we present an assay allowing
to measure and characterize remodelase activities in vitro and constructs made
to select for mistargetting of a GPI protein to the vacuole.