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

Cells grown to 70 to 90% confluence in 100-ram dishes were transfected by the calcium phosphate precipitation method (Graham and van der Eb, 1973) using 20 t~g of DNA per transfection. 36 h after transfection, cells were washed twice in PBS and incubated for 10 rain at 37~ in 15 ml of methionine-free DME containing 15 mM EDTA to release cells from the dish. Calls were then washed twice in methionine-free DME, and incubated for 30 min at 37"C in 2 ml of 0.5 mCi/ml [35S]methionine (Tran 35S-Label, ICN Radiochemicals, Irvine, CA) in methionine-free DME containing 5 % dialyzed FBS, 100 U/ml penicillin, and 100/~g/mi streptomycin. Pulselabeled ceils were chased for different periods at 370C in complete medium. Cell pellets were solubilized in 1 ml lysis buffer (0.5% [wt/vol] Triton X-100, 0.3 M NaC1, 50 mM Tris-HC1 buffer, pH 7.4). Specific proteins were isolated from detergent-solubilized cells and from culture supernatants using protein A-bound antibody to "lhc (7G7) as described (Bonifacino et al., 1990) . Proteins were resolved by one-dimensional SDS-PAGE under reducing conditions on 10% acrylamide gels. Quantitation was performed using a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA).