Linking synaptotagmin to vesicle fusion

Synaptotagmin 1 is an integral synaptic vesicle protein and is believed to be the major calcium sensor in synaptic vesicle fusion. Loss of synaptotagmin 1 from the nematode C. elegans results in an uncoordinated phenotype and decreased levels of neurotransmitter release. Synaptotagmin consists of two calcium/lipid binding C2 domains (C2A and C2B) separated by a short flexible linker (12 AA). While the role of the C2 domains has been extensively studied, the role of the short linker domain between them has not. It has been proposed that the linker sequence may be important in interacting with the SNAREs and with positioning the C2 domains in relation to each other and the SNARE complex. We explore two models for the role of the linker domain. 1) The sequence of the linker is important for function. To test the effects of the linker sequence the sequence was replaced with a flexible sequence consisting of repeats of glycine-glycine-serine (GGS). 2) The length of the linker is critical for its function. To test the effects of increasing the length of this linker domain the sequence was repeated, doubling the length. Initial results show partial rescue of endocytosis and exocytosis function for the flexible and double linker constructs. This suggests the sequence and length of the linker is important, but not vital, for synaptotagmin function. The next step is to determine whether endocytosis or exocytosis is being affected by these mutations.