Membrane fusion of secretory vesicles of the sea urchin egg in the absence of NSF.
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.
Whalley
Tim
T
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Timmers
Kim
K
Coorssen
Jens
J
Bezrukov
Ludmila
L
Kingsley
David H
DH
Zimmerberg
Joshua
J
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D003911:Dextrans / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D008561:Membrane Fusion / Q000187:drug effects; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D010063:Ovum / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012617:Sea Urchins / Q000166:cytology*; D022142:Secretory Vesicles / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013267:Stilbenes / Q000494:pharmacology; D013451:Sulfonic Acids / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000172:deficiency* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D003911:Dextrans; D013267:Stilbenes; D013451:Sulfonic Acids; D033921:Vesicular Transport Proteins; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; D005033:Ethylmaleimide