Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells.
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-alpha) is a zymogen granule (ZG) membrane protein characterized by an NH(2)-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-alpha in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-alpha truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-alpha(1-82), containing the J domain, significantly augmented Ca(2+)-stimulated amylase secretion. Effects of CSP-alpha(1-82) were concentration dependent, with a maximum 80% increase occurring at 200 microg/ml of protein. Although CSP-alpha(1-82) had no effects on basal secretion measured in the presence of < or =10 nM free Ca(2+), it did significantly augment GTP-gammaS-induced secretion under basal Ca(2+) conditions by approximately 25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca(2+)-stimulated secretion, implicating the CSP-alpha/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-alpha physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-alpha-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-alpha. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-alpha colocalization with VAMP 8. These data establish a role for CSP-alpha in regulating digestive enzyme secretion and suggest that CSP-alpha and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.
Weng
Ning
N
Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Baumler
Megan D
MD
Thomas
Diana D H
DD
Falkowski
Michelle A
MA
Swayne
Leigh Anne
LA
Braun
Janice E A
JE
Groblewski
Guy E
GE
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004798:Enzymes / Q000378:metabolism*; D005089:Exocytosis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D046790:Pancreas, Exocrine / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D055571:Secretory Pathway; D022142:Secretory Vesicles / Q000201:enzymology* / Q000378:metabolism
D004798:Enzymes; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D008565:Membrane Proteins; D010446:Peptide Fragments; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C491830:Vamp8 protein, rat; C088460:cysteine string protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium