Purification and analysis of TIP47 function in Rab9-dependent mannose 6-phosphate receptor trafficking

Abstract

TIP47 (tail interacting protein of 47 kDa) is a cytosolic protein that is essential for the transport of mannose 6-phosphate receptors (MPRs) from endosomes to the trans-Golgi. This protein is recruited from the cytosol onto the surface of late endosomes by Rab9 GTPase, which enables TIP47 to bind to MPR cytoplasmic domains with enhanced affinity. A mutation in a deep hydrophobic cleft of TIP47 (F(236)C) confers enhanced affinity binding to MPR cytoplasmic domains and stabilizes MPRs in living cells. We describe the purification of native and recombinant TIP47 proteins and assays that we use to monitor the function of this protein in MPR transport in living cells.