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Accumulation of a GPI‐Anchored Protein at the Cell Surface Requires Sorting at Multiple Intracellular Levels
Author(s) -
Grünfelder Christoph G.,
Engstler Markus,
Weise Frank,
Schwarz Heinz,
Stierhof YorkDieter,
Boshart Michael,
Overath Peter
Publication year - 2002
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1034/j.1600-0854.2002.30805.x
Subject(s) - endoplasmic reticulum , golgi apparatus , endosome , biology , microbiology and biotechnology , immunoelectron microscopy , glycoprotein , secretory pathway , intracellular , cell membrane , cell fractionation , exocytosis , membrane , biochemistry , immunohistochemistry , immunology
Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol‐anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady‐state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high‐pressure frozen and freeze‐substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo‐ and exocytosis in this organism, is 48–52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum → Golgi complex → cell surface) or three enrichment steps (endoplasmic reticulum → Golgi complex → endosomes → cell surface), suggesting sorting between several membrane compartments .