The Cysteine String Secretory Vesicle Protein Activates Hsc70 ATPase

doi:10.1074/jbc.271.42.25989

The Cysteine String Secretory Vesicle Protein Activates Hsc70 ATPase*

From the ^‡ Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305-5428 and the
^∥ Beckman Laboratories for Structural Biology, Department of Structural Biology, Stanford University Medical Center, Stanford, California 94305-5400

^¶ To whom correspondence should be addressed:
Dept. of Molecular and Cellular Physiology, Stanford University Medical Center, Stanford, CA 94305.
Tel.: 415-725-7545; Fax: 415-725-8021.

Abstract

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 (~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.

Footnotes

↵^§ The first two authors contributed equally to this work.
↵* This work was supported by National Institutes of Health Grant GM-39928. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CSP

cysteine string NSF

protein

N-ethylmaleimide-sensitive fusion protein

SNAP

oluble SF ttachment rotein

MOPS

4-morpholinepropanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

BisTris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
↵² S. M. Wilbanks and M. Souza, unpublished results.
↵³ J. E. A. Braun, unpublished results.
- Received July 16, 1996.
- Revision received August 8, 1996.