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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

Authors

  • Han W
  • Rhee JS
  • Maximov A
  • Lin W
  • Hammer RE
  • Rosenmund C
  • S├╝dhof TC

Journal

The Journal of biological chemistry

Publication Data

February 11, 2005

Associated Grants

None

Mesh Terms

  • 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