THE HSP70-COCHAPERONE, CYSTEINE STRING PROTEIN, INTERACTS WITH THE SNARE PROTEIN VAMP 8 AND REGULATES ACINAR CELL SECRETION

Abstract

We recently identified VAMP 8/endobrevin as an important zymogen granule SNARE protein that mediates acinar cell exocytosis. Investigation of potential protein interaction with VAMP 8 by coimmunoprecipitation, GST-pull-down assay and immunofluorescence microscopy revealed that VAMP 8 is present in a complex with a member of the DnaJ family: cysteine string protein (CSP). In contrast, the related pancreatic SNARE isoform VAMP 2 did not interact with CSP. In neurons, CSP acts as a cochaperone for the heat shock proteins HSC70 and HSP70 via interaction through its J-domain, which hydrolyses ATP and thereby activates HSC70 chaperone activity. In acini, CSP was found to interact with HSC70 under normal conditions and with HSP70 following its induction by heat shock. To identify a role for CSP in exocytosis, a CSP or a CSP truncation construct containing the J domain was introduced into perfringolysin O (PFO) permeabilized acinar cells and their effects on Ca2+-stimulated secretion was determined. In contrast to soluble constructs of VAMP 8, which inhibit exocytosis by 50%, CSP augmented acinar cell amylase secretion in a time and concentration-dependent manner, with an 80% maximum enhancement at 200 ug/ml of protein. These data demonstrate a specific role for CSP in mediating acinar cell secretion and moreover, implicate a role for HSC70 in modulating dynamic interactions of SNARE proteins during exocytosis.