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The intracellular location at which the G protein of vesicular stomatitis virus accumulated when transport was blocked at 20°C has been studied by biochemical, cytochemical, and immunocytochemical methods. Our results indicated that the viral G protein was blocked in that cisterna of the Golgi stack which stained for acid phosphatase. At 20°C this trans cisterna became structurally altered by the accumulation of G protein. This alteration was characterized by extensive areas of membrane buds which were covered by a cytoplasmic coat. These coated structures were of two kinds--those that labeled with anti-clathrin antibodies and those that did not. The clathrin-coated pits consistently did not label with anti-G antibodies. Upon warming infected cells to 32°C, G protein appeared on the surface within minutes. Concomitantly, the trans cisterna lost its characteristic structural organization. Double-labeling experiments were performed in which G protein localization was combined with staining for horseradish peroxidase, which had been taken up from the extracellular medium by endocytosis. The results suggest that the trans cisterna was distinct from the endosome compartment and that the latter was not an obligatory station in the route taken by G protein to the cell surface.

Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, O1, O4 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. O1 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. O4 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with O4 that had not yet begun to express the O1 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in O1-positive cells. Therefore, O4 stains, in addition to sulfatide and seminolipid, and unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing O4+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with O1, and thus R-mAb transiently stained a larger population of oligodendrocytes than did O1. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that O4, R-mAb, and O1 have multiple overlapping specificities, including previously unrecognized cross-reactions.

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