Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

Abstract

An assay has been developed for quantitating the reassociation of cortical secretory vesicles (CVs) with fragments of sea urchin egg plasma membrane attached to glass slides (PM lawns). Binding of S. pupuratus CVs to homologous PM lawns increased with time and CV concentration. The observation that CV binding was blocked by chymotrypsin digestion of the PM fragments suggested that a PM protein(s) is required for reassociation. The possibility that the extent of CV lysis that occurred during CV preparation (15.4 +/- 3.8% as assessed by ovoperoxidase assay) influenced reassociation was investigated by determining the effect of CV content proteins (isolated as fertilization product) on binding. Various concentrations of fertilization product (up to equivalent amounts of fertilization product and CV protein) had no effect on CV binding. The specificity of binding was investigated by assessing the ability of CVs to bind to PM lawns prepared from human red blood cells, and by determining the ability of heterologous vesicles to bind to egg PM fragments. PM lawns from HRBCs did not support CV binding; however, PM lawns prepared from the eggs of several species of sea urchin did bind S. pupuratus CVs. Vesicles from a partially purified preparation of yolk platelets bound to egg PM lawns with low efficiency (1/7 that of CVs), but immunofluorescence analysis with an anti-hyalin monoclonal antibody demonstrated that 74 +/- 9% of the bound vesicles were CVs that contaminated the yolk platelet preparation. Dioleoylphosphatidyl choline liposomes were also unable to bind to egg PM lawns. These results are consistent with hypothesis that CV binding to egg PM lawns is a specific, protein-mediated event.

Proteins on exocytic vesicles mediate calcium-triggered fusion.

“…Thus, the decrease in light scattering is a result of fusion. The calcium concentration required to trigger half maximal scattering change (Q30 uM) was similar to that reported for reconstituted cortical granule-to-PM fusion (CG-PM fusion) (10,11,18) and was higher than the Ca2+ sensitivity of exocytosis of intact planar cortices, 5 ,uM (Fig. 2).…”

“…However, the [Ca20+ in reconstituted CG-PM fusion is at least as high as 25 ,uM (10,11,18). Thus, the shift of Ca2" sensitivity is correlated with reconstitution itself.…”

“…While it is possible that trypsin might inhibit fusion indirectly by perturbing membrane structure, gross structural changes seem unlikely because digested granules do not lyse in the absence of Ca2+, even under the stress of centrifugation. The finding that, in reconstituted CG-PM systems, chymotrypsin treatment ofegg PMs prevents native cortical granule binding suggests that proteins tether granules onto the surface of the PM (18). Nonetheless, it is unlikely that the inhibition of vesicle fusion by trypsin is solely due to preventing CG-CG adhesion because (i) trypsin treatment of cortical granules enhances their aggregation (18), (ii) proteolysis does not remove cortical granules from isolated cortices (19), (iii) trypsin treatment did not stop the slow transfer ofDi-I from one granule in a clump to other granules, and (iv) treated granules did not fuse in the presence of Ca2+ even when they were pressed together by centrifugal force.…”

“…The finding that, in reconstituted CG-PM systems, chymotrypsin treatment ofegg PMs prevents native cortical granule binding suggests that proteins tether granules onto the surface of the PM (18). Nonetheless, it is unlikely that the inhibition of vesicle fusion by trypsin is solely due to preventing CG-CG adhesion because (i) trypsin treatment of cortical granules enhances their aggregation (18), (ii) proteolysis does not remove cortical granules from isolated cortices (19), (iii) trypsin treatment did not stop the slow transfer ofDi-I from one granule in a clump to other granules, and (iv) treated granules did not fuse in the presence of Ca2+ even when they were pressed together by centrifugal force. While it is possible that proteolytic fragments of proteins that are not involved in vesicle fusion might occlude essential sites of the fusion mechanism, the simplest interpretation of our data is that CG-CG fusion is mediated by a trypsin-sensitive protein that resides on cortical granules.…”

Reconstitution of Calcium-triggered Membrane Fusion Using “Reserve” Granules

“…It is possible that fraction C granules may be missing key components which may be required for biological docking but which are not required for fusion. This is supported by the observation that yolk granules attached to egg plasma membrane sheets with 1/7 the efficiency of isolated cortical granules in an in vitro binding assay (46). It is also possible that the components required for biological docking may be different from the components responding to the experimental manipulations used to bring granule membranes into close juxtaposition in this study.…”