Synthesis of Glycosyl-Phosphatidylinositol Anchors is Initiated in the Endoplasmic Reticulum

  • J. Vidugiriene
  • A. K. Menon
Conference paper
Part of the NATO ASI Series book series (volume 74)


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 [1]. 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 [3], 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 [4]. 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.


Endoplasmic Reticulum Endoplasmic Reticulum Marker Marker Enzyme Activity Post Nuclear Supernatant Eucaryotic Organism 
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Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • J. Vidugiriene
    • 1
  • A. K. Menon
    • 1
  1. 1.The Rockefeller UniversityNew YorkUSA

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