Accumulation of a GPI‐Anchored Protein at the Cell Surface Requires Sorting at Multiple Intracellular Levels

Abstract

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 glycosylphosphatidylinositolanchored 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 freezesubstituted 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 547 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.

The membrane-bound histidine acid phosphataseTbMBAP1 is essential for endocytosis and membrane recycling inTrypanosoma brucei

“…3C-E [see Fig. 1 and previous studies (Grünfelder et al, 2002;Grünfelder et al, 2003;Overath and Engstler, 2004) for relevant structures and details]. Specific immunolabelling was observed on endosomal cisternae ( Fig.…”

Characteristics of Novel Insect Defensin-Based Membrane-Disrupting Trypanocidal Peptides

“…In calculation of the relative surface PS density, the mode of PS fluorescence was divided by the average surface area (mm 2 ) of a single African trypanosome or pig erythrocyte (the control) based on published data. 21,22) Incubation of L-and D-type synthetic 9-mer peptides with human brain microvascular endothelial cells (HBMEC). To assess the biocompatibility and/or toxicity of the L-and D-type synthetic 9-mer peptides on host tissues, we used a well-described in vitro model of the human blood-brain barrier (BBB) composed of human brain microvascular endothelial cells (HBMEC).…”

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

“…on VSG trafficking, which have shown that the total VSG complement on the surface of T. brucei is internalized at enormous rate, resulting in a turnover time of 12 min (12,35,43). The fact that it takes more than an hour for the Tf-R to disappear from the surface after the addition of bovine Tf (Fig.…”

“…This is unexpected, as trypanosomes are normally able to handle the routing of new VSG efficiently. The Tf-R resembles a VSG dimer in structure and in its glycosylphosphatidylinositol anchor (23), but T. brucei makes at least 100-fold more VSG than Tf-R, and only 11% of this VSG is present intracellularly (12,35,43).…”