Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca 2+ -dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1's ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.synaptotagmin | Drosophila | synaptic vesicle | neurotransmitter release | exocytosis N eurotransmitter release requires temporal and spatial coupling of action potential-triggered Ca 2+ influx to synaptic vesicle fusion (1). The core fusion machine contains SNARE proteins found on the synaptic vesicle (v-SNAREs) and plasma membrane (t-SNAREs) that assemble into a four-helix bundle to bring the two bilayers into close apposition (2, 3). Besides SNAREs, Ca 2+ -binding proteins act to trigger release through fast synchronous and slow asynchronous pathways. Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that binds Ca 2+ and triggers synchronous vesicle fusion (4-9). Syt1 contains an intravesicular N-terminal tail, a single transmembrane segment, and a ∼60-residue linker that connects to two cytoplasmic Ca 2+ -binding C2 domains (10-13). Numerous Syt1 studies have focused on its cytoplasmic C2 domains, which interact with phospholipids and the SNARE complex in a Ca 2+ -dependent manner and are proposed to be the essential domains that trigger fusion (12, 14-21). In contrast, the significance of other structural elements of Syt1 remains poorly understood. Syt1 is predicted to facilitate synaptic vesicle fusion through a trans interaction with plasma membrane lipids (22-27). Tethering of Syt1 to synaptic vesicles through its transmembrane domain has been postulated to position the protein to properly target lipids and SNAREs, or to be required to generate force for pulling th...