Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

CV-I cells were plated at 60% confluence in 100-rnm tissue culture dishes and transfected with 20 pg of DNA by the calcium phosphate precipitation method. At 48 h after transfection, cells were trypsinized and replated in sterile 100-mm Petri dishes. At 60 h after transfection, cells were incubated for 5 to 10 rain in complete medium containing 10 mM Hepes/NaOH, pH 7.3, and 10 mM EDTA to release the cells into suspension. Cells were washed twice with ice-cold DME at 40C and incubated for 30 rain at 4~ with 125I-tabeled anti-Tac antibody at 1 x 107 cpm/ml ('~1 /~g/ml) as described (Weissman et al., 1986) . The labeled anti-Tac antibody was spun in a Beckman Airfuge (Beckman Instruments, Inc., Fullerton, CA) at 25 psi for 45 rain immediately before use to remove antibody aggregates. Cells were washed three times in ice-cold DME, resuspended in cold complete medium and incubated in a 37~ water bath to allow antibody uptake. At the indicated times, the cells were placed on ice, pelleted, and incubated in 250 ~,1 of DME with or without 1 mg/ml proteinase K for 30 min at 4"C. Cells were then centrifuged through a 200-#1 cushion of FBS. Radioactivity of cell pellets was measured in a Beckman 5500 gamma counter (Beckman Instruments, Inc.). All experiments were performed in duplicate.