Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule.
Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.
An
S J
SJ
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, United Kingdom.
Hansen
N J
NJ
Hodel
A
A
Jahn
R
R
Edwardson
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D004220:Disulfides / Q000032:analysis; D004792:Enzyme Precursors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D008566:Membranes / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry*; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D004220:Disulfides; D004792:Enzyme Precursors; D008565:Membrane Proteins; C107454:Sycn protein, rat; D014357:Trypsin