A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion
Zhuo Guan,
Maria Bykhovskaia,
Ramon A Jorquera,
Roger Bryan Sutton,
Yulia Akbergenova,
J Troy Littleton
Affiliations
Zhuo Guan
Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, United States
Maria Bykhovskaia
Department of Neurology, School of Medicine, Wayne State University, Detroit, United States
Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico
Roger Bryan Sutton
Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, United States
Yulia Akbergenova
Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, United States
Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, United States; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, United States
The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.
