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.


Sucrose Lymphoma Polypeptide Catalase Cavitation 


  1. 1.
    Cross, G.A.M. (1990) Glycolipid anchoring of plasma membrane proteins. Annu. Rev. Cell Biol. 6, 1 - 34.PubMedCrossRefGoogle Scholar
  2. 2.
    Doering, T.L., Masterson, W.J., Hart, G.W., and Englund, P.T. (1990) Biosynthesis of glycosylphosphatidylinositol membrane anchors. J. Biol. Chem. 265, 611 - 614.PubMedGoogle Scholar
  3. 3.
    Stevens, V.L., and Raetz, C.R.H. (1991) Defective glycosylphosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. J. Biol. Chem. 266, 10039 - 10042.PubMedGoogle Scholar
  4. 4.
    Storrie, B., and Madden, E. (1990) Isolation of subcellular organelles. Methods in Enzymol. 182, 203 - 235.CrossRefGoogle Scholar
  5. 5.
    Braell, W.A. (1988) Two sensitive, convenient, and widely applicable assays for marker enzyme activities specific to endoplasmic reticulum. Anal. Biochem. 170, 328 - 334.PubMedCrossRefGoogle Scholar

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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