Control of protein traffic between distinct plasma membrane domains. Requirement for a novel 108,000 protein in the fusion of transcytotic vesicles with the apical plasma membrane

Abstract

We have developed a cell-free system that reconstitutes the last step in transcytosis, i.e. the fusion of transcytotic transport vesicles with the apical plasma membrane (PM). Subcellular fractions containing transcytotic vesicles (the donor) or apical PM (the acceptor) were prepared from rat liver by sucrose density centrifugation. Fusion between the donor and acceptor fractions was measured by the conversion of the 120,000 transmembrane form of the polymeric IgA receptor (pIgA-R), an endogenous protein of transcytotic vesicles, to a processed fragment by a protease endogenous to the apical PM. Fusion occurred only at 37 degrees C and was critically dependent on the presence of ATP and cytosol. Fusion was inhibited by treating the in vitro fusion reaction with N-ethylmaleimide or by adding antibodies against N-ethylmaleimide-sensitive factor (NSF). We have previously identified a specific transcytotic vesicle-associated protein (TAP) and here show that TAP exists in both cytosolic and membrane-associated pools. Because of its exclusive interaction with transcytotic vesicles, we tested the involvement of TAP in distinct fusion processes. Removal of TAP inhibited fusion in an in vitro transcytotic fusion reaction but had no inhibitory effect in an in vitro endosome-endosome fusion system or in an in vitro intra-Golgi transport reaction. We propose that TAP represents part of the molecular machinery specifically involved in targeting and/or fusion of transcytotic vesicles with the apical PM.