TARGETING AND MISTARGETING OF PLASMA-MEMBRANE ADAPTERS IN-VITRO

Targeting and recruitment of the plasma membrane (PM) clathrin-coated vesicle adaptor complexes has been studied using an in vitro system ba sed on permeabilized acceptor cells and donor cytosol. Through the use of species- and/or tissue-specific antibodies, only newly recruited e xogenous PM adaptors are visualized. Targeting of PM adaptors can be s witched from the plasma membrane to a perinuclear compartment by GTPga mmaS or excess calcium. Prior treatment with brefeldin A prevents GTPg ammaS-induced mistargeting. Double-labeling immunofluorescence and imm unogold EM indicate that the perinuclear PM adaptor binding compartmen t is late endosomal. We propose that receptors for PM adaptors cycle b etween the plasma membrane and an endosomal storage compartment. Norma lly the receptors would be switched on only at the plasma membrane, bu t both GTPgammaS and calcium are capable of reversing this switch. Int racellular sequestration of PM adaptor receptors may provide the cell with a mechanism for up-regulating endocytosis following a burst of ex ocytosis.