The Journal of Cell Biology, Vol 119, 139-151, Copyright © 1992 by The Rockefeller University Press

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Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins

JC Hay and TF Martin
Program in Cell and Molecular Biology, University of Wisconsin, Madison 53706.

The biochemical events and components responsible for ATP-dependent Ca(2+)-activated secretion remain to be identified. To simplify the molecular dissection of regulated secretion, we have resolved norepinephrine (NE) secretion from semi-intact PC12 cells into two kinetically distinct stages, each of which was studied separately to discern its molecular requirements. The first stage consisted of MgATP- dependent priming of the secretory apparatus in the absence of Ca2+. MgATP-dependent priming was readily reversible and inhibited by a broad range of protein kinase inhibitors. The second stage consisted of Ca(2+)-triggered exocytosis which, in contrast to priming, occurred in the absence of MgATP. Both priming and triggering were found to be dependent upon or stimulated by cytosolic proteins. The priming and triggering activities of cytosol were functionally distinct as indicated by differing thermolability. Furthermore, active components in cytosol resolved by gel filtration were found to support either priming or triggering, but not both. For both priming and triggering reactions, several peaks of activity were detected; one of each type of factor was partially purified from rat brain cytosol, and found to be enriched for stage-specific activity. Two partially purified factors exhibiting stage-specific activity, a approximately 20-kD priming factor and approximately 300-kD triggering factor, were able to support regulated secretion as effectively as crude cytosol when used sequentially in the partial reactions. Further characterization of stage-specific cytosolic factors should clarify the nature of MgATP- and Ca(2+)-dependent events in the regulated secretory pathway.
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