Cell lysis induces redistribution of the GPI-anchored variant surface glycoprotein on both faces of the plasma membrane of Trypanosoma brucei

African trypanosomes are coated by 10 million copies of a single variant sp ecific glycoprotein (VSG) which are anchored in the plasma membrane by glyc osylphosphatidylinositol (GPI). A GPI-specific phospholipase C (GPI-PLC) tr iggers fast VSG release upon cell lysis but in vivo it is safely controlled and topologically concealed from its substrate by being intracellular, One enigmatic aspect of GPI-PLC action therefore consists of how it could gain access to the VSG in the exoplasmic leaflet of the membrane, The data pres ented herewith disclose an unexpected possible solution for this puzzle: up on cell rupture the VSG invades the cytoplasmic face of the plasma membrane which thus becomes double coated, This unusual VSG rearrangement was stabl e in ruptured plasma membrane from GPI-PLC null mutant trypanosomes but tra nsiently preceded VSG release in wild-type parasites, The formation of doub le coat membrane (DCM) was independent of the presence or activation of GPI -PLC, occurred both at 4 degrees C and 30 degrees C and was unaffected by t he classical inhibitor of VSG release, p-choromercuryphenylsulfonic acid (P CM), DCMs conserved the same coat thickness and association with subpellicu lar microtubules as in intact cells and were prone to form vesicles followi ng gradual detachment of the latter, Our data also demonstrate that: (i) GP I-PLC expressed by one trypanosome only targets its own plasma membrane, be ing unable to release VSG of another parasite; (ii) DCMs concomitantly form ed from trypanosomes expressing different VSGs do not intermix, an indicati on that DCM might be refractory to membrane fusion.