Interaction between Constitutively Expressed Heat Shock Protein, Hsc 70, and Cysteine String Protein Is Important for Cortical Granule Exocytosis in Xenopus Oocytes

Abstract

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 ϳ80%. 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 NH 2 -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.Eggs of many species exhibit cortical granule exocytosis, an early postfertilization event that leads to a sustained block of polyspermy (1). Although oocytes of Xenopus laevis must undergo maturation before sperm can elicit cortical granule exocytosis, immature oocytes (and eggs) of Xenopus secrete in response to activators of protein kinase C (2). Somewhat unexpectedly (given that the majority of regulated secretory events are initiated by an increase of cytosolic Ca 2ϩ , Refs. 3 and 4), cortical granule exocytosis in Xenopus oocytes is insensitive to changes of cytosolic Ca 2ϩ . For instance, this secretory event is not triggered by Ca 2ϩ ionophores (using physiological concentrations of Ca 2ϩ ); it cannot be evoked by direct injection of Ca 2ϩ into the cytoplasm, and it is unaffected by removal of extracellular Ca 2ϩ or by buffers that clamp cytosolic Ca 2ϩ below the resting level (5-7). Thus, oocytes offer an empirical avenue for assessing the function of proteins in a secretory pathway where calcium ions play no direct role. This issue is of considerable importance with respect to cysteine string proteins (csp 2 (s)), a class of proteins found associated with a broad range of regulated secretory organelles (8 -10).Csp was originally identified as a novel synaptic antigen in Drosophila (11), and several subsequent investigations indicated that csp might be important as a modulator of presynaptic Ca 2ϩ channels (12-17). However, it also became evident that regulation of presynaptic Ca 2ϩ channe...

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“…Of additional interest, the conserved ''J''-domain of CSP contains a motif, HPD, which is known to specifically bind to HSP70-like proteins and to stimulate HSP70 ATPase activity (the interaction of CSP with heat shock proteins is confined to HSP70/Hsc70, and it does not interact with other heat shock proteins such as HSP60 or HSP90) (10,11,45). In fact, interactions of CSP with HSP70 proteins have been suggested to play a role in exocytotic secretion (46)(47)(48); specifically CSP, in concert with HSP/HSC70-like proteins, may coordinate sequential protein-protein interactions to mediate various steps in the exocytotic process in synaptic vesicles (49), oocytes (47), and pancreatic b cells (50,51).…”

Larval excretory-secretory products from the parasite Schistosoma mansoni modulate HSP70 protein expression in defence cells of its snail host, Biomphalaria glabrata

“…The membrane was then blocked for 40 min at RT with 5% (w/v) non-fat dried milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 (TTBS), and incubated overnight at 4°C followed by 1 h at RT in primary anti-HSP70 monoclonal antibodies (BRM-22, Santa Cruz Biotechnology, Heidelberg, Germany, 1:1,000 in TTBS). This anti-HSP70 antibody has been used previously to detect HSP70/HSC70 in mammals and Xenopus, as well as homologues in invertebrates including the fruit fly, Drosophila melanogaster, and the zebra mussel, Dreissena polymorpha (Clayton et al 2000;Lilja et al 2007;Smith et al 2005).The membrane was then washed three times in TTBS before being incubated with an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000 in TTBS; Santa Cruz Biotechnology) for 3 h at RT. The immunoreactive signal was developed using SuperSignal West Pico chemiluminescent substrate (PerBioscience, Tatenhall, UK) and visualised using a GeneGnome chemiluminescence imaging system (Syngene, Cambridge, UK); band intensities were analysed using GeneTools software (Syngene).…”

A Role for Myosin 1e in Cortical Granule Exocytosis in Xenopus Oocytes

“…However, it is plausible that the apparent interaction between Myo1e and Csp goes beyond a stabilization of the membrane (lipid) binding of Myo1e. Recent investigations (20) indicate that Csp, in conjunction with a 70-kDa cognate heat shock protein (Hsc70), plays an important role in cortical granule exocytosis in oocytes. Thus, although the precise contribution of Csp (and Hsc70) to the exocytotic sequence remains to be clarified, the current experiments suggest that Csp function is likely to be intertwined with that of Myo1e, and possibly serve as a model for understanding the role of myosins in exocytosis.…”

“…Where indicated, each oocyte was extracted for immunoblot analysis of Myo1e or Myo1e constructs as described for csp in Ref. 20. For Fig.…”

“…The agarose beads were washed three times with buffer A and suspended in sample buffer for SDS-PAGE and immunoblot analysis as described in Ref. 20. Attempts to determine whether calmodulin co-immunoprecipitates with Myo1e were hampered by the lack of a suitable calmodulin antibody.…”