Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells

Abstract

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-α) is a zymogen granule (ZG) membrane protein characterized by an NH2-terminal “J domain” and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-α in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-α truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-α1-82, containing the J domain, significantly augmented Ca2+-stimulated amylase secretion. Effects of CSP-α1-82were concentration dependent, with a maximum 80% increase occurring at 200 μg/ml of protein. Although CSP-α1-82had no effects on basal secretion measured in the presence of ≤10 nM free Ca2+, it did significantly augment GTP-γS-induced secretion under basal Ca2+conditions by ∼25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca2+-stimulated secretion, implicating the CSP-α/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-α physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-α-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-α. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-α colocalization with VAMP 8. These data establish a role for CSP-α in regulating digestive enzyme secretion and suggest that CSP-α and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.