Reference: Schulz TA and Creutz CE (2004) The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family. Biochemistry 43(13):3987-95

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Abstract


The tricalbins are a recently discovered family of Saccharomyces cerevisae proteins containing a predicted N-terminal transmembrane domain and at least three C2 domains. They are thought to be yeast homologues of synaptotagmin, a hypothesis supported by structural similarities and past studies that implicated tricalbins in processes of membrane trafficking and sorting. We expressed and purified constructs consisting of single tricalbin C2 domains, and assayed their ability to bind lipids in response to calcium. Protein-lipid overlay assays indicated that the C-terminal C2 domains (C2C) of tricalbins 1 and 3 bind numerous species of acidic phospholipid, including phosphatidylserine and several phosphoinositides, and the amount of protein bound was greatly enhanced in the presence of 1 mM calcium. Sedimentation assays using mixed phosphatidylserine/phosphatidylcholine (PS/PC) vesicles confirmed that the C2C domains of tricalbin 1 and 3 bind membranes in a calcium-responsive manner and showed that they are more sensitive to calcium than the C2A domain of synaptotagmin I. Both assays revealed that all of the C2 domains of tricalbin 2 are insensitive to calcium. Fluorimetric assays exploiting the position of naturally occurring tryptophans in tricalbin 1 C2C and tricalbin 3 C2C confirmed that these domains are capable of binding calcium and that this is coupled to the binding of acidic phospholipid. Combining this with past protein-protein interaction data, we theorize that the calcium-insensitive tricalbin 2 mediates the creation of hetero-oligomeric tricalbin complexes in which tricalbin 1 or 3 or both supply a calcium-dependent membrane binding activity.

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Journal Article
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Schulz TA, Creutz CE
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