SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.
ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.
Kleene
R
R
Department of Cell Biology and Cell Pathology, Philipps University, Marburg, Germany.
Classen
B
B
Zdzieblo
J
J
Schrader
M
M
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D018909:src Homology Domains
D003432:Cross-Linking Reagents; D004792:Enzyme Precursors; C121683:Mta1 protein, rat; D011506:Proteins; D000681:Amylases