Functional Conservation of Cytosolic Proteins Required for Endosomal Vesicle Fusion

Recent studies suggest that intracellular membrane traffic relies upon families of related proteins which confer specificity to individual transport reactions but which operate in tandem with a ubiquitous fusogenic complex containing the N-ethylmaleimide-sensitive fusion protein (NSF). The extent to which components of this process are functionally conserved is apparent from the finding that yeast Sec18 protein (Sec18p) can substitute or mammalian NSF in intra-Golgi transport reactions. Here we report that yeast cytosol can support mammalian endosomal vesicle fusion, demonstrating conservation of cytosolic components required for this reaction. Furthermore, under conditions in which the fusion reaction is NSF-dependent we show that yeast Sec18p can functionally substitute for NSF, showing that the yeast protein is capable of catalysing at least two distinct mammalian membrane fusion events. In addition we exploit the complex pattern of sensitivity of the mammalian reaction to N-ethylmaleimide (NEM), coupled with the use of yeast cytosol, to dissect a number of factors required for fusion. We reveal at least three novel NEM-sensitive activities. One of these can be restored by yeast cytosol suggesting that it is functionally conserved.