Synthesis of Glycosyl-Phosphatidylinositol Anchors is Initiated in the Endoplasmic Reticulum
Numerous proteins from eucaryotic organisms are covalently modified by inositol-containing glycophospholipids (GPIs). Addition of a GPI anchor to protein occurs by cleavage of a carboxy-terminal signal sequence and attachment of a GPI precursor to the newly exposed α-carboxyl group of the polypeptide . The experiments described here are aimed at defining the sub-cellular localization of GPI synthesis. Since recent data from other laboratories indicated that GPI synthesis could be easily assayed in T cell (BW5147.3 thymoma) lysates , we chose to determine the intracellular location of GPI assembly by analyzing sub-cellular fractions from T cells. After disruption of the cells by nitrogen cavitation and removal of nuclei, 70–90% of the lysosomes (β-hexosaminidase activity) and peroxisomes (catalase activity) by low speed centrifugation, the post nuclear supernatant (PNS) was layered on a series of sucrose steps and centrifuged . Fractions were collected from the top of the tube and assayed for organelle-specific marker enzymes. As shown in Fig. 1, the endoplasmic reticulum (ER; dolichol-Pmannose synthase activity), Golgi (α-mannosidase II activity) and plasma membrane (PM; alkaline phosphodiesterase activity) were clearly separated.
KeywordsSucrose Lymphoma Polypeptide Catalase Cavitation