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J. Biol. Chem., Vol. 280, Issue 38, 32669-32675, September 23, 2005
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1
From the
Department of Molecular and Medical Pharmacology and
Departments of Neurobiology and Medicine, David P. Geffen UCLA School of Medicine, Los Angeles, California 90095
In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by
80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.
Received for publication, February 17, 2005 , and in revised form, July 25, 2005.
Note Added in ProofAn investigation of the role of the J-domain of csp in Drosophila appeared recently (Bronk, P., Nie, Z., Klose, M. K., Dawson-Scully, K., Zhang, J., Robertson, R. M., Atwood, H. L., and Zinsmaier, K. E. (2005) J. Neurosci. 25, 22042214).
* This work was supported by Grant NS31934 from the National Institutes of Health (to J. A. U.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed. Tel.: 310-825-3423; Fax: 310-206-8975; E-mail: cgundersen{at}mednet.ucla.edu.
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