Post-Golgi trafficking of the mannose 6-phosphate receptor in living cells

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1. Sorting of lysosomal enzymes is conducted by mannose 6-phosphate receptors (M6PR), which are transported between the trans-Golgi network (TGN), endosomes, and plasma membrane. In this research project, we examined functions of the extracytoplasmic domain of M6PR in its intracellular trafficking, and obtained the following conclusions.(1) Time-lapse imaging and FRAP (fluorescence recovery after photobleaching) analyses of GFP-M6PR, together with a chloroquine treatment suggest that the extracytoplasmic domain of M6PR is required for tight interaction with endocytic compartments and retention by them.(2) Experiments using a drug, U18666A, and fibroblasts derived from I-cell disease patients, suggest that the binding to the M6P ligands is involved in yet unidentified transport steps of M6PR. U18666A could be a useful tool to investigate new transport steps of M6PR.2. To investigate the function of clathrin adaptors, GGAs, that regulate the M6PR trafficking, we performed RNAi and dominant-negative experiments for GGA2. Although the depletion of GGA2 alone did not cause an apparent re distribution of M6PR, a lysosomal enzyme, cathepsin D, was significantly missecreted. These results suggest that missorting of lysosomal enzymes is not always accompanied by the drastic M6PR redistribution. We will perform detailed analyses as to which transport steps are regulated by GGAs.