Sidedness of Biosynthesis of Glycosylphosphatidylinositol Anchors in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Abstract

Many surface membrane glycoproteins of eucaryotes are attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthesis of these anchors proceeds through two stages. First, the synthesis of the protein and of a free glycosylphosphatidylinositol (GPI) is achieved separately. In a second step, the protein is hooked onto the preformed free GPI whereby a provisional C-terminal hydrophobic peptide is removed. The GPI-anchored protein is subsequently transported to the cell surface by way of vesicular traffic. It is presumed that the attachment of the preformed free GPI's to proteins occurs on the luminal surface of the endoplasmic reticulum (ER). The stepwise addition of sugars by glycosyltransferases onto phosphatidylinositol to form a free GPY is equally presumed to occur in the ER, but it is unclear whether these reactions take place at the cytosolic or the luminal side of the membrane. Here we tried to get some information on the membrane orientation of free GPYs in Saccharomyces cerevisiae surmising that their orientation might tell us something about the probable location of the biosynthetic process. When using trinitrobenzenesulfonic acid as a probe, we find that 75% of the free GPIs in intact ER-derived microsomes get derivatized, whereas 100% get derivatized in detergent-permeabihzed microsomes. This finding is compatible with the idea that in yeast lipid anchors are built up at the cytosolic surface.