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

Our in vivo data indicating that the M protein could acquire GalNAc in the absence of energy were supported by the biochemical analysis using SLO permeabilized cells. It is now generally accepted that for in vitro reconstitution of any vesicular transport step both cytosol and ATP are needed. Accordingly, in this study both cytosol and ATP were required for the newly synthesized M protein to acquire the Golgi modifications, consistent with the notion that ER to Golgi transport requires a vesicular transport step (Beckers and Balch, 1989; Beckers et al., 1990; Schekman, 1992) . In contrast, the intermediate compartment modification, GalNAc, was obtained in the presence of cytosol without ATP and in the presence of GTP3,S. In the absence of cytosol the results were more difficult to interpret. Under these conditions both ATP and UDP-GalNAc were required for efficient GalNAc addition. While the UDP-GalNAc requirement can be explained simply by the fact that this cytosolic precursor is lost after permeabilization, the ATP requirement is more difficult to explain. One obvious possibility relates to the phenomenon of"quality control" (Hurtley and Helenius, 1989) , the need for the protein to be folded in its correct conformation. The idea that ATP is not critical for folding of the M protein, which has only 25 amino acids exposed on the luminal side (Rottier et al., 1986) , is supported by our in vivo studies where ATP depletion had no obvious effect on the appearance of the M1 form. Whatever the explanation for the ATP requirement, we consider it unlikely that it reflects a vesicular transport step from the rough ER to the site of GalNAc addition since it occurs in the absence of cytosol and in the presence of GTPTS. In contrast, these conditions, as expected, blocked the appearance of the Golgi forms of the M protein.