C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1.
To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.
Pubmed ID: 15561725 RIS Download
Animals | Antigens, Thy-1 | Brain | Calcium | Calcium-Binding Proteins | Cell Nucleus | Electrophysiology | Green Fluorescent Proteins | Hippocampus | Immunohistochemistry | Immunoprecipitation | Liposomes | Membrane Glycoproteins | Mice | Mice, Knockout | Mice, Transgenic | Microscopy, Confocal | Models, Biological | Nerve Tissue Proteins | Neurons | PC12 Cells | Plasmids | Protein Binding | Protein Structure, Tertiary | Rats | Recombinant Fusion Proteins | SNARE Proteins | Semliki forest virus | Synapses | Synaptotagmin I | Synaptotagmins | Transfection | Transgenes | Vesicular Transport Proteins