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.
Han
Weiping
W
Center for Basic Neuroscience, the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
Rhee
Jeong-Seop
JS
Maximov
Anton
A
Lin
Weichun
W
Hammer
Robert E
RE
Rosenmund
Christian
C
Südhof
Thomas C
TC
D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D004594:Electrophysiology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008081:Liposomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050600:SNARE Proteins; D012672:Semliki forest virus / Q000235:genetics; D013569:Synapses / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins; D018800:Thy-1 Antigens / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D033921:Vesicular Transport Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; C485182:Cyan Fluorescent Protein; D008081:Liposomes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; C494312:Syt1 protein, rat; D018800:Thy-1 Antigens; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins; D002118:Calcium