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Cysteine string protein (CSP) is a 34 kDa secretory vesicle protein bearing a "J-domain" as well as a palmitoylated cysteine-rich "string" region used for membrane attachment. Mutation of the CSP gene causes impaired presynaptic neuromuscular transmission in Drosophila melanogaster, implicating CSP as part of the exocytotic protein machinery. The J-domain of CSP shares homology with the universally conserved DnaJ family, a group of proteins that act as co-chaperones with Hsc70 and its homologs. Hsc70 is an abundant neural protein with coupled protein binding and ATPase activities. We have investigated the CSP modulation of Hsc70 ATPase activity. Here we demonstrated that CSP enhances Hsc70 ATPase activity in a dose-dependent manner. CSP activation of Hsc70 was maximal ( approximately 12 times) at 1:1 stoichiometry and above. We show that a J-domain-containing fragment (amino acids 1-82) of CSP is sufficient for the activation of Hsc70. Neither CSP nor the amino-terminal fragment stimulate the activity of the isolated Hsc70 ATPase domain (amino acids 1-386). CSP does not significantly increase the activity of N-ethylmaleimide-sensitive fusion protein, another ATPase required for transport vesicle function. Our results suggest that CSP, a DnaJ family member associated with the secretory vesicle cycle regulates Hsc70 functions. Hsc70 may function within the biochemical pathways of exo- and endocytosis to promote the formation or dissociation of multimeric complexes or to regulate conformational changes.

The direct modulation of N-type calcium channels by G protein ␤␥ subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of Ntype channels and G␤␥ have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by G␤␥. When syntaxin 1A was coexpressed with (N-type) ␣ 1B ؉ ␣ 2 -␦ ؉ ␤ 1b channels in tsA-201 cells, the channels underwent a 18 mV negative shift in halfinactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein ␤␥ subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that G␤␥ physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with G␤␥ and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein ␤␥ subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.