THE N-ETHYLMALEIMIDE-SENSITIVE PROTEIN THIOL-GROUPS NECESSARY FOR SEA-URCHIN EGG CORTICAL-GRANULE EXOCYTOSIS ARE HIGHLY EXPOSED TO THE MEDIUM AND ARE REQUIRED FOR TRIGGERING BY CA2+
T. Whalley et A. Sokoloff, THE N-ETHYLMALEIMIDE-SENSITIVE PROTEIN THIOL-GROUPS NECESSARY FOR SEA-URCHIN EGG CORTICAL-GRANULE EXOCYTOSIS ARE HIGHLY EXPOSED TO THE MEDIUM AND ARE REQUIRED FOR TRIGGERING BY CA2+, Biochemical journal, 302, 1994, pp. 391-396
It is known that sea-urchin egg cortical-granule exocytosis is inhibit
ed by agents such as N-ethylmaleimide (NEM) which modify thiol groups.
The fusion-related proteins modified by these agents have yet to be i
dentified, nor is there information regarding the topography of these
thiol groups. Furthermore, the step in cortical-granule exocytosis at
which these thiol groups participate is unknown. In this study we have
investigated the topological properties of, and the temporal requirem
ent for the function of, the fusion-related thiol groups by treating t
he isolated exocytotic apparatus with high-molecular-mass dextrans and
BSA carrying thiol-reactive 3-(2-pyridyldithio)propionate groups. The
dextran derivatives inhibited exocytosis. The BSA derivative was much
less inhibitory. Inhibition was reversed by treatment with dithiothre
itol. When NEM was added to the dextran-derivative-treated exocytotic
apparatus, treatment with dithiothreitol completely reversed inhibitio
n, indicating that the dextran derivatives inhibit by reacting at the
NEM-sensitive sites. A pulse of Ca2+ applied in the presence of inhibi
tors did not trigger any fusion following the removal of the inhibitor
by dithiothreitol. These data show that the thiol groups, the modific
ation of which by NEM inhibits exocytosis, are exposed to the medium i
n terms of their accessibility to macromolecules. They also show that
the fusion-related thiol groups are required during the Ca2+-dependent
stage of exocytosis.