title: The glycosylphosphatidylinositol-PLC in Trypanosoma brucei forms a linear array on the exterior of the flagellar membrane before and after activation.
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.
authors.affiliation: School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
authors.forename: Orla
authors.initials: O
authors.lastname: Hanrahan
authors.forename: Helena
authors.initials: H
authors.lastname: Webb
authors.forename: Robert
authors.initials: R
authors.lastname: O'Byrne
authors.forename: Elaine
authors.initials: E
authors.lastname: Brabazon
authors.forename: Achim
authors.initials: A
authors.lastname: Treumann
authors.forename: Jack D
authors.initials: JD
authors.lastname: Sunter
authors.forename: Mark
authors.initials: M
authors.lastname: Carrington
authors.forename: H Paul
authors.initials: HP
authors.lastname: Voorheis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D005407:Flagella / Q000378:metabolism*; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D003902:Detergents; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C045519:paraflagellar rod proteins, Trypanosoma brucei; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase