Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which t...

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Bibliographic Details
Published in:The Journal of general physiology 2017-10, Vol.149 (10), p.921-934
Main Authors: Bohannon, Kevin P, Bittner, Mary A, Lawrence, Daniel A, Axelrod, Daniel, Holz, Ronald W
Format: Article
Language:English
Subjects:
Animals
Cattle
Cell Membrane - metabolism
Cells, Cultured
Chemical reactions
Chromaffin cells
Chromaffin Cells - metabolism
Chromaffin Cells - ultrastructure
Granular materials
Humans
Hydrogen-Ion Concentration
Immunocytochemistry
Membrane Fusion
Microscopy
pH effects
Physiology
Plasminogen Activator Inhibitor 1 - metabolism
Polyamide-imides
Proteases
Protein Binding
Proteins
Secretory Vesicles - metabolism
Secretory Vesicles - ultrastructure
t-Plasminogen activator
Tissue Plasminogen Activator - metabolism
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Summary:A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species.
ISSN:0022-1295
1540-7748
DOI:10.1085/jgp.201711842