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

Abstract

Ca 2+ -triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca 2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca 2+ sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr 2+ , a weak Ca 2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca 2+ , suggesting that IA acts at the Ca 2+ -sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…Settle CV-CV fusion assays revealed no differences between fresh and incubated eggs, indicating that CV were able to form critical inter-membrane attachments necessary for a robust fusion response (Fig. 2b) [8,26,27,31,38,39,72,84,97]. Overlapping activity curves and kinetic responses found for both CV-CV and CV-PM fusion ( Fig.…”

“…The following parameters were analyzed: (1) ability to raise fertilization envelopes upon Ca 2+ influx (native exocytosis); (2) triggered CV-PM fusion in CSC preparations (exocytosis in vitro); (3) triggered CV-CV fusion in vitro; and (4) the CV membrane proteome and (5) (Fig. 2a), and initial fusion rate was 73.8±2.6%/s, comparable to freshly isolated CV [26,27,38,72]. Settle CV-CV fusion assays revealed no differences between fresh and incubated eggs, indicating that CV were able to form critical inter-membrane attachments necessary for a robust fusion response (Fig.…”

“…Removing/blocking membrane PE also inhibited initial fusion kinetics. As efficiency parameters are linked to integrity of CHOL-rich microdomains [26,27,38,72], yet PE does not actively support domain formation/stability, our data provide clues as to the physical nature of microdomains and the localized, functional domain composition at fusion sites [25]. Simply, (1) different negative curvature lipids may contribute to a given fusion site, and (2) highly localized and somewhat more fluid PE regions within the broader microdomain matrix may be critical to PFM/FFM (see [25], Fig.…”

Lipid Dynamics in Exocytosis

“…In contrast to the consistent inhibition of fusion with a variety of thiol reactive reagents, a particular startling finding has been that the simple alkylating reagent iodoacetamide promotes the Ca 2? sensitivity and kinetics of triggered release (Furber et al 2009a(Furber et al , b, 2010. The site of action appears to be at or near a Ca 2?…”

Dissecting the mechanism of Ca2+‐triggered membrane fusion: Probing protein function using thiol reactivity

“…The conserved nature of Ca 2+ ‐triggered membrane fusion 1,2 allows for the use of simplified in vitro model systems to study the underlying molecular mechanisms. Isolated secretory vesicles (cortical vesicles; CV) from sea urchin oocytes undergo fusion with the plasma membrane, 3–8 other CV 9–16 and artificial bilayers 17,18 in response to an increase in [Ca 2+ ] free . Unlike other types of secretory vesicles that tend to rapidly deprime during isolation, CV remain fully primed and fusion ready, not requiring ATP or any other cytosolic factors for fusion to occur 3,7 .…”