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 ( 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.
Highlights
Regulated secretion from neural, endocrine, and exocrine cells takes place via a series of discrete steps, including docking, fusion, and recycling of transport vesicles
We conclude that the ATPase activity of Hsc70 is strongly regulated by the vesicle protein Cysteine string protein (CSP)
To test the specificity of the Hsc70/CSP interaction, we investigated the effects of various recombinant proteins on Hsc70 ATPase activity
Summary
Proteins and Other Reagents—Preparation of the peptide fluoresceinFYQLALT was described by Takeda and McKay [29]. The fusion proteins recovered by binding of the glutathione S-transferase domain to glutathione-agarose beads (Sigma) were resuspended in 10 mM MOPS (pH 7.0), 4.5 mM Mg(CH3COO) 150 mM KCl and incubated twice in 5 mM ATP at 4 °C for 20 min. The column was washed sequentially with 75 mM, 1 M, and 75 mM KCl. Protein was eluted with 75 mM KCl, 3 mM ATP. Hsc70-containing fractions were dialyzed against 25 mM BisTris (pH 6.3) and loaded onto a Pharmacia Mono P column equilibrated with the same buffer, and proteins were eluted with 10% Polybuffer 74 (Pharmacia Biotech Inc.) (pH 4.0). The latest eluting peak from the isoelectric focusing column was loaded onto Superdex 75 column and developed in 40 mM Hepes, 150 mM KCl, 4.5 mM MgCl2 (pH 7). ATP hydrolysis rates were computed as linear fits to ADP produced versus time
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