The C2B domain of synaptotagmin I is a Ca2+-binding module

Josep Ubach, Ye Lao, Imma Fernandez, Demet Arac, Thomas C. Südhof, Jose Rizo-Rey

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Abstract

Synaptotagmin I is a synaptic vesicle protein that contains two C2 domains and acts as a Ca2+ sensor in neurotransmitter release. The Ca2+-binding properties of the synaptotagmin I C2A domain have been well characterized, but those of the C2B domain are unclear. The C2B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca2+-dependent manner, leading to an attractive model whereby Ca2+-dependent multimerization of synaptotagmin I via the C2B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca2+-dependent C2B domain dimerization, as well as in analyses of other C2B domain interactions. To shed light on these issues, the C2B domain has now been studied using biophysical techniques. The recombinant C2B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C2B domain interactions, including Ca2+-dependent dimerization. NMR experiments show that the pure recombinant C2B domain binds Ca2+ directly but does not dimerize upon Ca2+ binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C2A and C2B domains, participates in a high molecular weight complex as a function of Ca2+. These results show that the recombinant C2B domain of synaptotagmin I is a monomeric, autonomously folded Ca2+-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C2B domains or requires a posttranslational modification.

Original languageEnglish (US)
Pages (from-to)5854-5860
Number of pages7
JournalBiochemistry
Volume40
Issue number20
DOIs
StatePublished - May 22 2001

ASJC Scopus subject areas

  • Biochemistry

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    Ubach, J., Lao, Y., Fernandez, I., Arac, D., Südhof, T. C., & Rizo-Rey, J. (2001). The C2B domain of synaptotagmin I is a Ca2+-binding module. Biochemistry, 40(20), 5854-5860. https://doi.org/10.1021/bi010340c