Fc εRI-mediated hydrolysis of phosphoinositides in permeable membrane vesicles

Abstract

We have used hypotonic lysis of cytoplasts derived from rat basophilic leukemia (RBL) cells to prepare orgenelle and cytoplasm-depleted membrame vesicles called ‘RBL cell ghosts’ (Dreskin and Metzger, 1991). Unlike other membrane preparations, the RBL ghosts hydrolyze phosphoinositides (PIs) in response to aggregation of the high affinity receptor for IgE (Fc εRI) and have proven useful for studies of the molecular events involved in transduction of this signal. A significant limitation of these preparations is that they are sealed. Thus, to incorporate membrane-impermeant molecules (such as ATP) into the intravesicular space of the ghosts, they must be added as the ghosts are formed. We have now overcome this problem by permeabilizing the ghosts with α-toxin from S. aureus and find that, following permeabilization, ghosts activated via Fc εRI, hydrolyze PIs for a longer time than do non-permeabilized ghosts. As in the intact ghosts, this response is absolutely dependent upon ATP and is enhanced by the addition of either phosphoenolpyruvate (PEP) or creatine phosphate (CP). This report demonstrate that we can now man3pulate the intravesicular environment of the RBL ghosts and extends the utility of these preparations as a model system for the study of signal transduction following activation via Fc εRI.