Fusion Rapidly Follows Vesicle Transport to the Target Membrane in Protein Transport through the Golgi Apparatus in Vitro. A Re-evaluation of Transport Kinetics Based on the Finding that the Glycosylation Used to Mark Transport, and Not Transport Itself, is Rate Limiting in the Assay
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A well characterized assay measuring protein transport between compartments of the Golgi apparatus (W.E. Balch et al, 1984, Cell 39:405416) utilizes glycosylation of a membrane protein to mark transit. Previously, kinetic analysis of the assay relied on the assumption that the glycosylation that is used to mark transport occurs very rapidly upon fusion of transport vesicles with their target membranes. Here we report that, in fact, glycosylation is rate limiting in this assay and therefore that fusion proceeds substantially faster than previously thought. This finding alters the previous model of transport which held that there was a lengthy interval after vesicles attach to their target before fusion occurs. Our findings indicate that a presumptive pre-fusion intermediate, the “NEM resistant intermediate” is an artifact of the kinetic experiments used to define it.
KeywordsGolgi Apparatus Transport Vesicle Vesicle Fusion Sugar Nucleotide Transport Assay
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