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Title: Molecular characterization of syncollin, a novel syntaxin-binding protein
Author: An, S. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Exocytosis in the pancreatic acinar cell involves the fusion of zymogen granules (ZGs) with the apical domain of the plasma membrane, and the subsequent release of digestive enzymes into the acinar lumen. Although SNARE proteins have been implicated in many membrane trafficking steps, it is not known if these and other components of the "membrane fusion machine" are required for exocytosis in the exocrine pancreas. The aim of this research has been to investigate the biochemical properties of a novel syntaxin-binding protein, syncollin, that is contained in the membranes of ZGs. Syncollin has a molecular mass of 16 kDa and is predicted to have β sheet-forming regions. When solubilized in TX-100, syncollin binds to syntaxin at low Ca2+ concentrations and dissociates at concentrations known to stimulate exocytosis. The nature of this interaction is direct - as purified recombinant syncollin behaves similarly to the native protein from detergent-solubilized extracts of ZG membranes - and specific: syncollin binds to only syntaxins 1A and 2, the latter isoform's localization being restricted to the apical plasma membrane in acinar cells. Recombinant syncollin inhibits fusion in vitro between ZGs and pancreatic plasma membranes, and its potency falls as Ca2+ concentration rises. Because syncollin is also present on the granules of the parotid gland, it was initially suggested that syncollin acts as a Ca2+- sensitive regulator of exocytosis in exocrine tissues. Subsequent studies of syncollin's biochemistry, however, have led to a reappraisal of this proposed role. Syncollin possesses intramolecular disulfide bonds which allow it to resist denaturation by SDS and boiling temperatures. Reduction of the disulfide bonds with DTT does not result in dramatic unfolding of conformation, but it does affect conformation. Crosslinking experiments reveal that syncollin may form homo-oligomers in situ. However, TX-100-solubilized syncollin sediments as a monomer when subjected to sucrose density gradients. When the anionic detergent taurodeoxycholate (TDoC) is used in lieu of TX-100, syncollin is able to sediment as a ˜150-kDa complex. Additional experiments favouring the use of TDoC include the following: syncollin is highly resistant to proteases in situ and in TDoC, but not in TX-100; and DoC-solubilized syncollin is still capable of binding syntaxin 2, but not to syntaxin 1A. Syncollin is tightly associated with the lipid bilayer and can be released only at high pH. This observation served to establish a two-step purification protocol for the native protein. At pH 11, syncollin monomerizes but can spontaneously homo-oligomerize into the ˜150-kDa complex once purified (at natural pH) and solubilized in TDoC. These and other characteristics of syncollin are reminiscent of certain pore-forming polypeptides and proteins. A modified role for syncollin in enzyme secretion is discussed in light of these new findings.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available