Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a -propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K ؉ -induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.Synaptic vesicle fusion and the subsequent release of neurotransmitter require the formation of heterotrimeric SNARE 2 complexes formed from plasma membrane proteins syntaxin1A and SNAP-25 (Q-SNAREs) with the synaptic vesicle membrane protein VAMP/synaptobrevin (R-SNARE) (1-3). Present on opposing membranes, these SNAREs combine and engage in thermodynamically stable coiled-coil interactions that bridge the two membranes and catalyze their fusion (4). The formation of SNARE complexes is spatially and temporally controlled by accessory components that lend additional specificity to SNARE pairing, arrest SNARE complex intermediates, and/or lower the energy required for fusion (4 -6). Ultimately, it is the functional activity of these regulators on SNARE complex assembly that determines the dynamics of the exocytotic event.Tomosyn is an important regulator of SNARE complex formation whose mechanism of action remains unclear. Initially identified in neurons (7-8), tomosyn, a soluble R-SNARE protein, was considered to be a negative effector of fusogenic SNARE complex assembly through interactions with syntaxin1A and SNAP-25 that preclude the binding of VAMP2,...
Priming is the process by which vesicles become available for fusion at nerve terminals and it is modulated by numerous proteins and second messengers. One of the prominent members of this diverse family is Tomosyn. Tomosyn has been identified as a Syntaxin-binding protein; it inhibits vesicle priming, but its mode of action is not fully understood. Tomosyn's inhibitory activity depends on its N-terminal WD40-repeat domain and is regulated by the binding of its SNARE-motif to Syntaxin. The present review describes new physiological information on Tomosyn's function and addresses possible interpretations of these results in the framework of the recently described crystal structure of the yeast Tomosyn homolog Sro7. We also present possible molecular scenarios for vesicle priming and the involvement of Tomosyn in these processes. KeywordsTomosyn; Syntaxin; Priming; SNARE; Synaptic Transmission Vesicle priming as a key process in synaptic transmissionSynaptic transmission at the nerve terminal involves several steps that lead to the timely and controlled release of neurotransmitter. Following the arrival and docking of a vesicle at the plasma membrane (PM), it undergoes a series of maturation steps that render it fusioncompetent. These steps are collectively known as the vesicle priming process and are crucial for vesicle fusion. Priming is coordinated by a series of protein-protein interactions that occur between cytosolic, vesicular and PM proteins [1][2][3]. A key step in vesicle priming is the formation of multiple heterotrimeric SNARE complexes between the PM SNARE proteins Syntaxin1a and SNAP-25 and the vesicular protein Synaptobrevin/VAMP2 [4][5][6][7][8]. Full assembly of the SNARE complex is thought to bring the vesicle into close apposition with the PM and may catalyze the fusion reaction [9]. The priming process is highly regulated by several accessory proteins, such as Munc18, Munc13, Tomosyn, Rabphilin and Complexin. Changes in the levels of some synaptic proteins alter specific steps in synaptic transmission and affect synaptic plasticity processes [10][11][12][13][14]. All of these proteins interact with at least one member of the SNARE protein family. The purpose of this review is to discuss the priming process and the effect of Tomosyn on vesicle priming and synaptic transmission. TomosynTomosyn was first discovered as a Syntaxin1a-binding protein in a pulldown assay from rat cerebral cytosol and accordingly, received its Japanese name: tomo ("friend" in Japanese) of syn (Syntaxin) [15]. It is expressed in the brain and colocalizes with Syntaxin in synapse- [15,17,18] and other organelles such as insulin-containing granules [19,20], and it also localizes to the PM through its interaction with Syntaxin [21,22]. Tomosyn has three distinct domains ( Fig. 1): a C-terminal region containing an R-SNARE, Synaptobrevin-like coiled-coil domain; an N-terminal region enriched with WD40 repeats that are predicted to fold into a propeller-like structure, and a hypervariable linker domain that differs b...
Olfactory associative learning in Drosophila is mediated by synaptic plasticity between the Kenyon cells of the mushroom body and their output neurons. Both Kenyon cells and their inputs from projection neurons are cholinergic, yet little is known about the physiological function of muscarinic acetylcholine receptors in learning in adult flies. Here, we show that aversive olfactory learning in adult flies requires type A muscarinic acetylcholine receptors (mAChR-A), particularly in the gamma subtype of Kenyon cells. mAChR-A inhibits odor responses and is localized in Kenyon cell dendrites. Moreover, mAChR-A knockdown impairs the learning-associated depression of odor responses in a mushroom body output neuron. Our results suggest that mAChR-A function in Kenyon cell dendrites is required for synaptic plasticity between Kenyon cells and their output neurons.
Background: Tomosyn's WD40 domain affects its ability to inhibit exocytosis. Results: Unstructured loops in the WD40 domain are involved in tomosyn's diffusion and organization on the plasma membrane. Conclusion: These key loops mediate tomosyn's binding to the SNARE protein SNAP25. Significance: Novel findings regarding tomosyn's membranal distribution and interactions shed new light on regulation of exocytosis by the SNARE complex and tomosyn.
The protein tomosyn decreases synaptic transmission and release probability of vesicles, and is essential for modulating synaptic transmission in neurons. In this study, we provide a detailed description of the expression and localization patterns of tomosyn1 and tomosyn2 in the subareas of the mouse hippocampus. Using confocal and two-photon high-resolution microscopy we demonstrate that tomosyn colocalizes with several pre- and postsynaptic markers and is found mainly in glutamatergic synapses. Specifically, we show that tomosyn1 is differentially distributed in the mouse hippocampus and concentrated mainly in the hilus and mossy fibers. Surprisingly, we found that tomosyn2 is expressed in the subiculum, CA1 and CA2 pyramidal cell bodies, dendrites and spines, and colocalizes with PSD95, suggesting a postsynaptic role. These results suggest that in addition to the well-characterized presynaptic function of tomosyn in neurotransmitter release, tomosyn2 might have a postsynaptic function, and place tomosyn as a more general regulator of synaptic transmission and plasticity.
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