Abstract
The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca(2+)-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-gamma-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca(2+)-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.
Highlights
Cortical vesicles (CVs) are protein-filled secretory vesicles found in most animal eggs (Peres and Bernardini, 1985; Chandler, 1991; Tahara et al, 1996)
In addition to fusion with the plasma membrane (PM), isolated CVs can fuse with each other (Vogel and Zimmerberg, 1992) in response to micromolar concentrations of Ca2+, showing that the vesicles themselves possess the entire machinery required for Ca2+-dependent membrane fusion
CV exocytosis is unaffected by non-hydrolyzable ATP analogs
Summary
Cortical vesicles (CVs) are protein-filled secretory vesicles found in most animal eggs (Peres and Bernardini, 1985; Chandler, 1991; Tahara et al, 1996). The fully primed CVs exist, already docked to the plasma membrane (PM), and disruption of unfertilized eggs in Ca2+-free medium produces sheets of plasma membrane with the CVs firmly attached (Vacquier, 1975; Zimmerberg et al, 1985). In such an egg cortex preparation, exocytosis is triggered by the addition of micromolar [Ca2+]free (Baker and Whitaker, 1978; Sasaki and Epel, 1983; Zimmerberg and Liu, 1988; Blank et al, 1998), and this ionic signal is the only requirement for fusion in vitro (Whitaker, 1987). Characterization of the mechanism of CV-CV fusion has shown that it is the same as for exocytosis in vitro (Coorssen et al, 1998)
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