A novel assay reveals a role for soluble N-ethylmaleimide-sensitive fusion attachment protein in mannose 6-phosphate receptor transport from endosomes to the trans Golgi network

Abstract

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (α-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and α-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that α-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with α-SNAP, NSF can increase the extent of transport. These data show that α-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.