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J. Cell Biol.,
Volume 143, Number 7, December 28, 1998 1831-1844
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.
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