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

We and others have shown that the M protein of MHV acquires O-linked sugars posttranslationally (Tooze et al., The incubations were followed by a mAb anti-biotin, a rabbit serum to mouse IgG and protein A. 1988; Krijnse-Locker et al., 1992a) . The maturation of the O-linked oligosaccharides of the M protein follows a distinct pattern; in pulse-chase studies up to five different forms of the M protein appear sequentially and can be resolved on SDS gels (see Fig. 13 A) . To be able to relate the results obtained in earlier studies using sac(-) ceils with the L cells preferred for the morphological experiments in the present study, we compared the pattern of O-glycosylation of the M protein in both cells (Fig. 13 B) . To visualize the different forms of the M protein, cells were labeled at 5 h after infection continuously for 3 h, with [35S]methionine. In agreement with earlier data (Tooze et al., 1988; Krijnse-Locker et al., 1992a) in sac(-) cells five different forms were apparent (see Fig. 13 A for a summary). In L ceils the pattern of O-glycosylation was slightly different. The M5 form appeared to be absent while the M~ and M3 bands were more pronounced. The identity of the M protein bands in L cells was confirmed using biotinylated lectins. When we used Helix pomatia, which recognizes GalNAc, to detect the M protein, the Mt form was clearly enriched (Fig. 13 B) . The lectin also precipitated some of the M3 form, but not M0 and M4. In contrast, wheat germ agglutinin (WGA) preferentially recognized the M3 and M4 forms, consistent with the notion that this lectin is specific for SA (as well as for N-acetyl-glucosamine, which is probably not present in the M protein). The specificity of HPA was confirmed by adding 10 mM GalNAc at the start of the lectin incubation, which caused the disappearance of both the M~ and M3 bands, while GalNAc had no such effect on the WGA precipitation.