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+.
It is known that sea-urchin egg cortical-granule exocytosis is inhibited by agents such as N-ethylmaleimide (NEM) which modify thiol groups. The fusion-related proteins modified by these agents have yet to be identified, 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 requirement for the function of, the fusion-related thiol groups by treating the 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 dithiothreitol. When NEM was added to the dextran-derivative-treated exocytotic apparatus, treatment with dithiothreitol completely reversed inhibition, indicating that the dextran derivatives inhibit by reacting at the NEM-sensitive sites. A pulse of Ca2+ applied in the presence of inhibitors did not trigger any fusion following the removal of the inhibitor by dithiothreitol. These data show that the thiol groups, the modification of which by NEM inhibits exocytosis, are exposed to the medium in terms of their accessibility to macromolecules. They also show that the fusion-related thiol groups are required during the Ca(2+)-dependent stage of exocytosis.
Whalley
T
T
Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Sokoloff
A
A
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D003911:Dextrans / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects*; D005260:Female; D008565:Membrane Proteins / Q000378:metabolism; D010063:Ovum / Q000187:drug effects / Q000378:metabolism*; D011247:Pregnancy; D012617:Sea Urchins; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D003911:Dextrans; D008565:Membrane Proteins; D013388:Succinimides; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D005033:Ethylmaleimide; D002118:Calcium