Determinants of GPI-PLC localisation to the flagellum and access to GPI-anchored substrates in trypanosomes

In Trypanosoma brucei, glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor that releases variant surface glycoprotein (VSG) from dying cells. In live cells, GPI-PLC is localised to the plasma membrane where it is concentrated on the flagellar membrane, so activity or access...

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Veröffentlicht in:PLoS pathogens 2013-08, Vol.9 (8), p.e1003566-e1003566
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description In Trypanosoma brucei, glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor that releases variant surface glycoprotein (VSG) from dying cells. In live cells, GPI-PLC is localised to the plasma membrane where it is concentrated on the flagellar membrane, so activity or access must be tightly regulated as very little VSG is shed. Little is known about regulation except that acylation within a short internal motif containing three cysteines is necessary for GPI-PLC to access VSG in dying cells. Here, GPI-PLC mutants have been analysed both for subcellular localisation and for the ability to release VSG from dying cells. Two sequence determinants necessary for concentration on the flagellar membrane were identified. First, all three cysteines are required for full concentration on the flagellar membrane. Mutants with two cysteines localise predominantly to the plasma membrane but lose some of their flagellar concentration, while mutants with one cysteine are mainly localised to membranes between the nucleus and flagellar pocket. Second, a proline residue close to the C-terminus, and distant from the acylated cysteines, is necessary for concentration on the flagellar membrane. The localisation of GPI-PLC to the plasma but not flagellar membrane is necessary for access to the VSG in dying cells. Cellular structures necessary for concentration on the flagellar membrane were identified by depletion of components. Disruption of the flagellar pocket collar caused loss of concentration whereas detachment of the flagellum from the cell body after disruption of the flagellar attachment zone did not. Thus, targeting to the flagellar membrane requires: a titratable level of acylation, a motif including a proline, and a functional flagellar pocket. These results provide an insight into how the segregation of flagellar membrane proteins from those present in the flagellar pocket and cell body membranes is achieved.
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subjects Animals
Biology
Cell Line
Cell Membrane - enzymology
Cell Membrane - genetics
Flagella (Microbiology)
Flagella - enzymology
Flagella - genetics
Glycosylphosphatidylinositol Diacylglycerol-Lyase - genetics
Glycosylphosphatidylinositol Diacylglycerol-Lyase - metabolism
Health aspects
Host-parasite relationships
Inositol
Mice
Mice, Knockout
Mutation
Oligosaccharides - genetics
Oligosaccharides - metabolism
Phospholipases
Physiological aspects
Plasma
Plasmids
Proteins
Protozoan Proteins - genetics
Protozoan Proteins - metabolism
Trypanosoma
Trypanosoma brucei brucei - enzymology
Trypanosoma brucei brucei - genetics
title Determinants of GPI-PLC localisation to the flagellum and access to GPI-anchored substrates in trypanosomes
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