Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein.
After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.
D000818:Animals; D000953:Antigens, Protozoan; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003080:Cold Temperature; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma
D000953:Antigens, Protozoan; D006023:Glycoproteins; D008565:Membrane Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D014415:Tunicamycin; D008985:Monensin; D010738:Type C Phospholipases