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 contrast to the HPA, the M protein could be detected, using antibodies against both the NH2 terminus and the COOH terminus, in both the rough ER and the budding compartment (Fig. 7) , as well as in the Golgi stack (not shown). The concentration of the M protein in the budding compartment was, however, significantly higher than in the rough ER (Fig. 7, C and D) . The labeling with the COOH-terminal antibodies was significantly increased in sections of cells treated with SLO before fixation, presumably because the cytoplasmic epitopes became more accessible. Figure 9 . Localization ofrab 2 in SLO-permeabilized MHV infected L cells. In A the labeling (arrows) is seen on tubular-reticular membranes on one side of the Golgi stack (G). Labeling is also evident next to a budding virion (arrowhead). In B, a higher magnification area is shown of the labeling associated with the budding compartment (arrowheads, budding virions). C shows extensive labeling of the tubulo-cisternal membranes (star) that appear very electron dense in the Epon sections (see Fig. 3 A, asterisk) . The arrowhead indicates rab 2 labeling close to a budding virion. Bars, 200 nm. Figure 10 . Labeled cryo-sections of MHV-infected cells after SLO permeabilization and GTP3~S treatment. A and B shows a single labeling with anti B-cop (arrows). Note the large proliferation of B-cop reactive buds/vesicles around the Golgi stack (G) and adjacent to the nucleus (N). The arrowheads indicate budding or budded virions that are enclosed within membrane structures that have B-cop reactive buds/vesicles on their periphery. The B-cop reactive vesicles are easily distinguishable from the putative clathrin coated vesicles (C). F denotes putative intermediate filaments that often are more easily seen after extracting the cytosol. In C, the section was double labeled with B-cop (10 nm gold) and Helix pomatia (HPA, 6 nm gold, small arrows). The cop vesicles and buds are dearly evident around the Golgi stack (G) and the large arrow indicates a copreactive bud that is closely apposed to, and possibly continuous with the rough ER. The HPA labeling colocalizes to a large extent with the B-cop reactive vesicles (double arrow). The large arrowhead shows a budding virion on one side of the Golgi stack, closely adjacent to B-cop reactive vesicles. C, putative clathrin coated vesicles. Bars, 100 nm. Figure 11 . Double-labeled cryo-sections of MHVinfected L cells treated with SLO without (A) or with (B) GTP~S treatment. A is double labeled for PDI (10 nm gold) and the M protein (NH2-terminal antibodies, 6 nm gold). Note the small amount of labeling for PDI (indicated) that we consistently see over the virion-containing structures (strongly labeled for the M protein) on one side of the Golgi stack (G). InB, the PDI (10 nm) is double labeled with anti-E-cop (6 rim). PDI is evident in the rough ER which is directly continuous with the membranes containing H-cop reactive buds/vesicles. In this particular example no PDI labeling is seen over the H-cop reactive region to the right of the figure. Bars, 100 nm.