TRANSPORT OF A LYSOSOMAL MEMBRANE GLYCOPROTEIN FROM THE GOLGI TO ENDOSOMES AND LYSOSOMES VIA THE CELL-SURFACE IN AFRICAN TRYPANOSOMES

gp57/42 is a membrane glycoprotein localized in the trans-Golgi, flage llar pocket region of the cell surface, endosomes and lysosomes of blo odstream forms of Trypanosoma brucei rhodesiense, Pulse-chase immunopr ecipitation experiments revealed that gp57/42 acquires a unique N-link ed oligosaccharide recognized by the CB1 monoclonal antibody 20-30 min utes after protein synthesis, probably in the trans-Golgi. We refer to gp57/42 molecules that carry the CB1 eptiope as CB1-gp, Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecip itates, Since p57 and p42 share 10 of 13 methionyl peptides, we conclu de that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhib ited when cells were chased in two thiol protease inhibitors or in 3,4 -diiso-coumarin, but was inhibited by leupeptin. Cell surface biotinyl ation was used to determine if newly synthesized CB1-gp was transporte d from the Golgi to the surface. When cells were pulse labeled and cha sed for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes , suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accu mulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 mi nutes at 37 degrees C, the biotinylated CB1-gp contained both p57 and p42, The p57 in biotinylated CB1-gp was not cleaved to p42 if the addi tional incubation was done at 4 or 12 degrees C. This suggests that tr ansport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature, When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1 -gp, suggesting that CB1-gp molecules had passed through the processin g compartment and then appeared on the cell surface. Thus, a major por tion of the newly synthesized CB1-gp is routed from the Golgi to endoc ytic compartments via the cell surface. In trypanosomes this process i nvolves a unique surface domain, the flagellar pocket. Otherwise, this pathway of lysosomal membrane glycoprotein transport has been conserv ed in evolution from the trypanosomes to the vertebrates.