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

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment pro tein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events, A lthough NSF and alpha-SNAP have been shown to be required for many mem brane transport processes, their role in the transport of mannose 6-ph osphate receptors from endosomes to the trans Golgi network was not es tablished. We present here a novel in vitro assay that monitors the tr ansport of cation-dependent mannose 6-phosphate receptors between endo somes and the trans Golgi network, The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transp ort of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we sho w that alpha-SNAP strongly stimulates transport in reactions containin g limiting amounts of cytosol. Together with alpha-SNAP, NSF can incre ase the extent of transport. These data show that alpha-SNAP, a solubl e component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.