A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

For analysis of G proteins, HeLa cells (4 h postinfection) or COS-7 cells (44 h posttransfection) were incubated in cysteine-free medium for 10 min and then labeled for 30 min in 0.5 ml cysteine-free medium containing 50 ACi ["S]cysteine. Cells were harvested immediately, or after various times of chase as above. Cells were lysed as above, and G proteins immunoprecipitated with either 3 Al of a polyclonal rabbit anti VSV serum, 3 Al of a rabbit anti-peptide serum which recognizes the G cytoplasmic tail (27) , or with 2 jI of mAbs Il or 114 (23) . To show that Gml spanned the membrane, HeLa cells were labeled for 10 min, scraped from the dish, dounced 50 times with a tight-fitting pestle, and treated with or without 100 jig/ml TPCK-trypsin (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 60 min at 0°C. PMSF was added to 50 mM, microsomes were solubilized in detergent solution as above, and G proteins immunoprecipitated with the polyclonal antiVSV serum .