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

In the present study we have used mouse hepatitis virus as a tool to investigate the ER-Golgi boundary. MHV was chosen, because extensive studies by Tooze et al. (1984 Tooze et al. ( , 1985 Tooze et al. ( , 1988 argued strongly that this virus buds into membrane structures on the cis side of the Golgi complex and that the M (El) protein of the virus acquires the first of its O-linked Figure 16 . Pulse-chase analysis of the M protein in SLOpermeabilized cells. Infected L cells were pulse labeled (p) for 2 min at 6 h after infection. SLO was allowed to bind on ice for 10 min. After removal of excess SLO, cells were permeabilized by incubation for 4 min at 37°C. Cells were incubated for 30 min on ice to deplete the cytosol. Supernatant was replaced by SLO-buffer containing an ATP-depleting system (ATP, -) or an ATPregenerating system (ATP, +) and the labeled proteins chased (c) for 60 min at 37°C. To test the effect of various compounds on the GalNAc addition, the following reagents were added: 50 /zM GTP'yS, 100 #M UDP-GalNAc, except in the indicated case where 1 mM was added, and cytosol at 3.5 mg/ml. The second lane (-SLO) from the left shows the chase in intact cells.