Abstract
Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-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 alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.
Highlights
When N-ethylmaleimide-sensitive fusion protein (NSF) is mixed together with ␣-SNAP and a detergent extract of salt-washed Golgi membranes, NSF and ␣-SNAP become incorporated into a 20 S complex [7]
We study the transport of mannose 6-phosphate receptors (MPRs) between endosomes and the trans Golgi network (TGN), both in cultured cells
We were unable to determine if NSF was required for endosome-to-TGN transport because this step requires a factor that is more sensitive to N-ethylmaleimide than NSF [22]
Summary
GDI and Rab91⁄7GDI complexes were prepared as described [23]. Mouse cation-dependent mannose 6-phosphate receptor (MPR46) cDNA was a gift of Dr Bernhard Hoflack (EMBL, Heidelberg, Germany). The bacterial expression plasmid pMW183 containing His6::NodQ2 was a gift of Dr Sharon Long (Stanford University). Dr James Rothman (Sloan-Kettering, New York), Dr Sidney Whiteheart (Kentucky College of Medicine, Lexington), and Dr Thomas Mayer (University of Muenster, Germany) kindly provided expression plasmids for His-tagged versions of NSF, ␣-SNAP, and Rab1b N121I, respectively. The following chemicals (Sigma) were kept as stocks: 100 ϫ protease inhibitor mix: 1 mg/ml leupeptin, 4 mg/ml aprotinin, 100 M pepstatin A, stored at Ϫ80 °C; 20 mg/ml cycloheximide in ethanol and 20 mM primaquine in H2O, stored at Ϫ20 °C
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