Mild proteolytic digestion restores exocytotic activity to N- ethylmaleimide-inactivated cell surface complex from sea urchin eggs

Abstract

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of StrongyIocentrotus purpuratus increased the free Ca 2+ concentration required to elicit exocytosis. Although a small increase in the Ca 2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca 2+ (0.5 raM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). in this report we demonstrate that NEM inhibits by increasing the Ca 2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca 2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.Cell surface complex (CSC) ~ prepared from the eggs of the sea urchin Strongylcocentrotus purpuratus is comprised of the vitelline layer, the plasma membrane and attached cortical secretory vesicles (CVs), also referred to as cortical granules (1). In response to physiologically relevant concentrations of free Ca 2+ the CSC undergoes a reaction that morphologically approximates exocytosis (2-4). This response can be followed microscopically (2-5), enzymatically (6, 7), and turbidimetAbbreviations used in this paper: CSC, cell surface complex; CV, cortical vesicle; DFP, diisopropylfluorophosphate; NEM, N-ethylmaleimide; PKME, buffer made with PIPES, KCI, MgCI2, and EGTA; SBT1, soybean trypsin inhibitor.rically (6,8). Recent investigations have shown that exocytosis in this in vitro model system can be inhibited by phenothiazine drugs (6,7,9) and by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM; reference 6). Inhibition by sulfhydryl-modifying reagents suggested that proteinaceous elements of the CSC were required for cortical exocytosis and prompted us to investigate the effect of proteolytic digestion of CSC and NEM-inhibited CSC on cortical exocytosis. We show that cortical exocytosis can be completely inhibited by prolonged pro...

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

“…Using the stage-specific urchin CSC and CV preparations, previous studies have shown that thiol-reactive reagents inhibit fusion by blocking free sulfhydryl group(s) on proteins [6,[11][12][13][18][19][20][21][22]. In contrast, we now show that treatment with the thiol reagent IA has a biphasic effect on membrane fusion.…”

“…Notably, increasing incubation time up to 1 h never resulted in the return of Ca 2+ sensitivity to control values nor inhibition of the extent of fusion, even with 300 mM IA (data not shown). These results suggest that IA targets thiol group(s) affecting the efficiency of the membrane fusion reaction but that it does not effectively access sites that interfere with the ability of CV to fuse with the target membrane, as is seen with NEM [6,[11][12][13][18][19][20][21][22].…”

“…In earlier efforts to modify the release mechanism, the thiol-reactive reagent, N-ethylmaleimide (NEM), was shown to inhibit membrane fusion in both the CV-PM [18][19][20][21][22] and CV-CV assays [6,[11][12][13]. NEM inhibits both Ca 2+ sensitivity and the ability to fuse in a dosedependent manner; treatment with 5 mM NEM consistently leaves CV irreversibly fusion incompetent.…”

Proteins on exocytic vesicles mediate calcium-triggered fusion.

“…Protein Modifiers Inhibit CG-CG. CG-PM fusion can be inhibited by trypsin treatment of isolated cortical fragments, suggesting that proteins sensitive to trypsin are involved in the cortical reaction (19). We found that trypsin treatment of granules, under conditions similar to that which inhibits exocytosis in cortices, prevented them from fusing with each other, despite the presence of =350 AM calcium (Table 2).…”

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…Specific interactions between proteins in the CG membrane and proteins in the plasma membrane mediate vesicle docking and fusion (Jackson et al, 1985;Vater and Jackson, 1989). In egg activation of sea urchins, it is likely that these interactions occur through the SNARE complex of proteins (Conner et al, 1997;Conner and Wessel, 2001;Leguia and Wessel, 2004).…”