Abstract
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.
Highlights
The external cell surface of the mammalian bloodstream form of African trypanosomes is covered with a densely packed protein coat predominantly composed of a single polypeptide species, the variant surface glycoprotein (VSG) [1,2]
African trypanosomes are unicellular parasites with a single flagellum that maintain a persistent infection through antigenic variation based on changes in a densely packed cell surface coat of variant surface glycoprotein (VSG)
We have investigated the relationship between this subcellular localisation and VSG shedding ability of the glycosylphosphatidylinositol phospholipase C (GPI-PLC)
Summary
The external cell surface of the mammalian bloodstream form of African trypanosomes is covered with a densely packed protein coat predominantly composed of a single polypeptide species, the variant surface glycoprotein (VSG) [1,2]. Bloodstream form trypanosomes contain a GPI-phospholipase C (GPIPLC) [8,9,10] and when plasma membrane integrity is compromised, for example by hypotonic lysis, hydrolysis by GPI-PLC releases all VSG from the plasma membrane within five minutes [10,11]. In a population of cells, the half-life of VSG is ,30 hours, equivalent to several cell generations [12,13], indicating that there is very tight regulation of GPI-PLC activity and/or access to VSG in live cells
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