title: Vesicle fusion in protein transport through the Golgi in vitro does not involve long-lived prefusion intermediates. A reassessment of the kinetics of transport as measured by glycosylation.
abstract: The well-characterized cell-free assay measuring protein transport between compartments of the Golgi [Balch, W. E., Dunphy, W. G., Braell, W. A., & Rothman, J. E. (1984) Cell 39, 405-416] utilizes glycosylation of a glycoprotein to mark movement of that protein from one Golgi compartment to the next. Glycosylation had been thought to occur immediately after vesicles carrying the glycoprotein fuse with their transport target. Therefore, the kinetics of glycosylation were taken to reflect the kinetics of vesicle fusion. We previously isolated and raised monoclonal antibodies against a protein (the prefusion operating protein, POP) which is required in this assay at a step after vesicles have apparently been formed and interacted with the target membranes, but long before glycosylation takes place. This was therefore presumed to be a reaction involving targeted but unfused vesicles. Here we report that POP is identical to uridine monophosphokinase, as revealed by molecular cloning. We show that POP is not active in transport per se but instead enhances the glycosylation used to mark transport. This indicated that, contrary to previous assumptions, glycosylation might lag significantly behind vesicle fusion. We directly show this to be true. This alters the interpretation of several earlier studies. In particular, the previously reported existence of a late, prefusion intermediate, the "NEM-resistant intermediate", can be seen to be due to effects on glycosylation and not indicative of true fusion events.
authors.affiliation: Cell Biology Unit, Upjohn Company, Kalamazoo, Michigan 49007.
authors.forename: R R
authors.initials: RR
authors.lastname: Hiebsch
authors.forename: B W
authors.initials: BW
authors.lastname: Wattenberg
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D014169:Transformation, Bacterial; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D000255:Adenosine Triphosphate; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase