Abstract
Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.
Highlights
The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is present in bloodstream forms of T. brucei [1,2,3] and in many other eukaryotic cells
The glycosylphosphatidylinositol-PLC only occurs in the bloodstream form, where it removes the surface coat after it enters the tsetse fly vector
All approaches consistently show that the GPI-PLC is located exclusively in the outer leaflet of the plasma membrane covering the flagellum, where it is confined to a narrow linear array adjacent to the flagellar attachment zone
Summary
The GPI-PLC is present in bloodstream forms of T. brucei [1,2,3] and in many other eukaryotic cells. The enzyme cleaves the GPI-anchor of the VSG and other GPI-anchored proteins, provided the 2-position on the inositol is underivatized, forming free diacylglycerol in the membrane and, probably, a 1,2-cyclic phosphate on the inositol ring, which remains attached to the released VSG [4,5]. This cleavage converts the membrane-bound form of the VSG (mVSG) to the soluble released form of the VSG (sVSG) [6]. Catalytic activity itself does not require Ca2+ [1,3]
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