Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

Pulse-labeled ceils were rinsed on ice twice with PBS and once with SLObuffer containing 100 t~g/ml cycloheximide, 2 mM methionine, I mM DTT. The cells were incubated on ice for 10 rain with 2 U/ml SLO in SLO-buffer with the above mentioned components. The cells were rinsed twice with icecold SLO-buffer and were permeabilized by a 4-min incubation at 37"C. The cells were then again put on ice in SLO-buffer containing 2 mM CaCO3 and 4 mM EGTA but without DTT, and cytosol allowed to leak out for 30 min. The cells were rinsed twice with SLO-buffer and labeled proteins chased for 60 min in SLO-buffer/Ca-EGTA containing, in addition to cycloheximide and methionine, an ATP-regenerating (1 mM ATE 8 mM creatinine phosphate, 50 t~g/ml creatinine kinase) or an ATP-depleting (37.5 U/ml hexokinase, 12.5 mM glucose) system. When indicated 50/xM GTPTS (Boehrioger GmbH), 100 t~M or 1 mM UDP-GalNAc (Sigma Chemical Co.) were added. Cytosol was made from Hela spinner cells as before (van der Sluijs et al., 1990) and was added to the permeabilized cells at a final protein concentration of 3.5 mg/rnl.