Advertisement

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)

Abstract

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.

Keywords

Endoplasmic Reticulum Endoplasmic Reticulum Marker Marker Enzyme Activity Post Nuclear Supernatant Eucaryotic Organism 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  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

Personalised recommendations