N-ethylmaleimide-sensitive protein(s) involved in cortical exocytosis in the sea urchin egg: localization to both cortical vesicles and plasma membrane.
The exocytotic release of secretory products from fragments of sea urchin egg cortex has been shown to be inhibited by covalent modification of membrane sulfhydryl groups with N-ethylmaleimide (NEM). Exocytotically competent preparations of reconstituted cortex, formed by recombination of purified cortical vesicles (CVs) with fragments of egg plasma membrane (PM) were also inhibited by treatment with NEM. The cellular localization of sulfhydryl-containing constituent(s) responsible for inhibition was investigated by treating CVs and/or PM with NEM prior to reconstitution. Both native cortex and cortex reconstituted with NEM-treated components were challenged with calcium-containing buffers. Exocytosis was monitored by phase-contrast microscopy, and quantitated by light scattering. Evidence for CV-PM fusion was obtained with an immunofluorescence-based assay that permits visualization of the transport of CV content proteins across the PM. Cortex reconstituted by recombination of NEM-treated CVs with untreated PM or by recombination of untreated CVs with NEM-treated PM was exocytotically competent, whereas cortex formed by recombination of NEM-treated CVs with NEM-treated PM was inactive. These results: (1) support the hypothesis that the mechanism of exocytosis in native and reconstituted cortex is the same; (2) provide evidence that both CV and plasma membranes participate in the release of CV contents from reconstituted cortex; and (3) suggest that sulfhydryl-containing protein(s) present on the surface of purified CVs and plasma membrane are involved in exocytosis.
Jackson
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RC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03756.
Modern
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PA
D000818:Animals; D002118:Calcium; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D010063:Ovum / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D012617:Sea Urchins; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011506:Proteins; D013438:Sulfhydryl Compounds; D005033:Ethylmaleimide; D002118:Calcium