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Vol. 17, Issue 12, 5211-5226, December 2006

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C₂B Polylysine Motif of Synaptotagmin Facilitates a Ca²⁺-independent Stage of Synaptic Vesicle Priming In Vivo

Carin A. Loewen^*, Soo-Min Lee, Yeon-Kyun Shin, and Noreen E. Reist^*

*Molecular, Cellular, and Integrative Neuroscience Program, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523; and Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011

Submitted July 24, 2006; Revised September 6, 2006; Accepted September 7, 2006
Monitoring Editor: Jeffrey Brodsky

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca²⁺ affinity of release, and, at low Ca²⁺, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca²⁺ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt^null mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca²⁺-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca²⁺-independent docking and/or priming of synaptic vesicles in vivo.

Address correspondence to: Noreen E. Reist (reist{at}lamar.colostate.edu)

Abbreviations used: t-SNARE heterodimers, syntaxin/SNAP-25; v-SNARE, VAMP or synaptobrevin; SNARE complex, VAMP/syntaxin/SNAP-25; minis, miniature synaptic potentials.

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