Reciprocal Intramolecular Interactions of Tomosyn Control Its Inhibitory Activity on SNARE Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of tomosyn on the SNARE complex formation. The repressed inhibitory activity of tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.Synaptic vesicles are transported to the presynaptic plasma membrane where Ca²⁺ channels are located. Depolarization induces Ca²⁺ influx into the cytosol of nerve terminals through the Ca²⁺ channels, and this Ca²⁺ influx initiates the fusion of the vesicles with the plasma membrane, finally leading to exocytosis of neurotransmitters (1). Soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP)² receptors (SNAREs) are essential for synaptic vesicle exocytosis (2-5). Synaptic vesicles are endowed with vesicle-associated membrane protein 2 (VAMP-2) as a vesicular SNARE, whereas the presynaptic plasma membrane is endowed with syntaxin-1 and SNAP-25 as target SNAREs. VAMP-2 interacts with SNAP-25 and syntaxin-1 to form a stable SNARE complex (6-9). The formation of the SNARE complex then brings synaptic vesicles and the plasma membrane into close apposition, and provides the energy that drives the mixing of the two lipid bilayers (3-5, 9).Tomosyn is a syntaxin-1-binding protein that we originally identified (10). Tomosyn contains N-terminal WD40 repeats, a tail domain, and a C-terminal domain homologous to VAMP-2. The C-terminal VAMP-like domain (VLD) of tomosyn acts as a SNARE domain that competes with VAMP-2. Indeed, a structural study of the VLD revealed that the VLD, syntaxin-1, and SNAP-25 assemble into a SNARE complex-like structure (referred to as tomosyn complex hereafter) (11). Tomosyn inhibits SNARE complex formation by sequestering t-SNAREs through the tomosyn complex formation, and thereby inhibits SNARE-dependent neurotransmitter release. The large N-terminal region of tomosyn shares similarity to the Drosophila tumor suppressor lethal giant larvae (Lgl), the mammalian homologues M-Lgl1 and M-Lgl2, and yeast proteins Sro7p and Sro77p (12, 13). Consistent with the function of tomosyn, Lgl family members play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane in yeast and epithelial cells (12, 13). However, only tomosyn, Sro7, and Sro77 have the tail domains and the VLDs, suggesting that their structural regulation is evolutionally conserved. Recently, the crystal structure of Sro7 was solved and revealed that the tail domain of Sro7 binds its WD40 repeats (14). Sec9, a yeast counterpart of SNAP-25, also binds the WD40 repeats of Sro7. This binding inhibits the SNARE complex formation and exocytosis by sequestering Sec9. In addition, binding of the tail domain to the WD40 repeats causes a conformational change of Sro7 and prevents the interaction of the WD40 repeats with Sec9, leading to regulation of the inhibitory activity of Sro7 on the SNARE complex formation (14). However, the solved structure of Sro7 lacks its VLD. Therefore, involvement of the activity of the VLD in the conformational change of Sro7 remains elusive.Genetic studies in Caenorhabditis elegans showed that TOM-1, an ortholog of vertebrate tomosyn, inhibits the priming of synaptic vesicles, and that this priming is modulated by the balance between TOM-1 and UNC-13 (15, 16). Tomosyn was also shown to be involved in inhibition of the exocytosis of dense core granules in adrenal chromaffin cells and PC12 cells (17, 18). Thus, evidence is accumulating that tomosyn acts as a negative regulator for formation of the SNARE complex, thereby inhibiting various vesicle fusion events. However, the precise molecular mechanism regulating the inhibitory action of tomosyn has yet to be elucidated.In the present study, we show that the tail domain of tomosyn binds both the WD40 repeats and the VLD and functions as a regulator for the inhibitory activity of tomosyn on the SNARE complex formation. Our results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain serves for controlling the inhibitory activity of the VLD.