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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01077

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Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF

Tim Whalley^1,2,*, Kim Timmers^1,*, Jens Coorssen³, Ludmila Bezrukov¹, David H. Kingsley⁴ and Joshua Zimmerberg^1,

¹ Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² Centre for Extracellular Matrix Biology, Department of Biological Sciences, University of Stirling, Stirling, FK9 4LA, UK
³ Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
⁴ United States Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, W.W. Baker Center, Delaware State University, Dover, DE 19901, USA

Author for correspondence (e-mail: joshz{at}helix.nih.gov)

Accepted 5 January 2004

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 Ca²⁺-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--S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca²⁺-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

Key words: Sea urchin, Egg, Exocytosis, Fusion, Cortical vesicles

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