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© The Rockefeller University Press, 0021-9525/1998//1831 $5.00
The Journal of Cell Biology, Volume 143, Number 7, , 1998 1831-1844

Regular Articles

Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay



Sylvie Urbé, Lesley J. Page, and Sharon A. Tooze

Secretory Pathways Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.

Key Words: fusion • immature secretory granule • prohormone convertase 2 • secretogranin • NSF

Abbreviations used in this paper: CCV, clathrin-coated vesicle; ISG, immature secretory granules; MP, membrane pellet; MPR, mannose-6-phosphate receptor; MSG, mature secretory granule; NEM, N-ethylmaleimide; NSF, N-ethylmalemide–sensitive fusion protein; PC2, prohormone convertase 2; PNS, postnuclear supernatant; SgII, secretogranin II; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; STI, soybean trypsin inhibitor; t-SNARE, target SNARE; v-SNARE, vesicle SNARE.

S. Urbé's present address is Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L34BU, UK.


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