Pulse-chase experiments using L-[35S]tnethionine suggest that Trypanosoma brucei MITat 1.2 variable surface glycoprotein (VSG) synthesized in the rough endoplasmic reticulum, a process that takes 6-8 min, is shuttled to the Golgi complex 8 min later. Labeling of ultrathin frozen sections with affinity-purified anti-cross-reacting determinant (CRD) IgG followed by protein A-colloidal gold shows that the CRD is localized in the trans-Golgi region. cis-Golgi is not labeled. VSG, when solubilized by treatment with the detergent Nonidet P-40, behaves on sucrose density gradients as a non-membrane protein with a sedimentation value of 5 S. In contrast, VSG solubilized in the presence of Zwittergent TM 3-14 yielded several VSG-containing fractions >5 S, and only the 5S fraction contained the CRD. Lack of the CRD in VSG complexes with sedimentation values >5 S suggests that this determinant is either masked from antibody, perhaps by involvement in polymer formation, or represents the membrane form African trypanosomes have on their surface a continuous 12-to 15-nm-thick glycoprotein coat (VSG) (1) that has been shown to vary both immunochemically (2)(3)(4)(5) and biochemically (1, 5-8) during different stages of host infection. It is known that the VSGs of both Trypanosoma brucei and Trypanosoma congolense contain at least two classes of carbohydrate side chains (9). One, referred to as the internal side chain, is located in the COOH-terminal third of the glycoprotein, contains mannose and N-acetylglucosamine, and can be removed by endo-f-N-acetylglucosaminiidase (1Q). Its incorporation itito VSG is mediated by a dolichol monophosphate intermediate (9) and is inhibited by the antibiotic tunicamycin (11)(12)(13)(14). Recently, using highly enriched Golgi and endoplasmic reticulum (ER) fractions isolated from African trypanosomes (15, 16), we tentatively concluded that inhibition of N-acetylglucosaminyl transferase by tunicamycin Al occurred only in the smooth ER subfraction, suggesting that core glycosylation of the VSG may occur in this organelle (16) and not in the rough ER, as previously assumed (10,11,13). As with any centrifugally prepared smooth ER subfraction, it is difficult to ascertain the ratio of ER to other vesiculated fragments of other membranous organelles. Studies showing the in vivo incorporation of mannose into VSG also suggest that N-glycosylation occurs subsequent to VSG synthesis (14). The existence of a dolichol-dependent mechanism in the smooth ER of trypanosomes is not at all unlikely, especially in light of recent evidence by Rip et al. (17), which shows that in rat liver significant quantities of dolichol phosphate are found in both smooth ER and Golgi. Of more potential pharmacological interest is the second class of carbohydrate side chain, which contains mannose, N-acetylglucosamine, and galactose (9), and which is located at the COOH-terminal end of the protein conjugated through an ethanolamine linkage to the a-carboxyl group (18), possibly via phosphatidylethanolamine (19). The a...