Friends and foes in synaptic transmission: the role of tomosyn in vesicle priming

Abstract

Priming is the process by which vesicles become available for fusion at nerve terminals and 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. The inhibitory activity of tomosyn depends on its N-terminal WD40-repeat domain and is regulated by the binding of its SNARE motif to syntaxin. Here, we describe new physiological information on the function of tomosyn and address 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.

Figure 1. Domain structure of Tomosyn and its homologs

The N-terminus of all Tomosyn isoforms is enriched with WD40 repeats that are predicted to fold into an N-terminal propeller-like structure and a C-terminal propeller-like structure (light green boxes). The C-terminus of all Tomosyn isoforms contains an R-SNARE, Synaptobrevin-like coiled-coil domain (red box). Mouse m-Tomosyn is shown as a representative of all Tomosyn isoforms. A SNARE-like domain exists in Sro7 and in L(2)gl (dark blue box). A hypervariable domain (HVR) is part of the putative C-terminal propeller-like structure (dark green box) and varies in length between the different splice variants (only the m-Tomosyn isoform is shown here). The PKA phosphorylation site in Tomosyn and the 3 aPKC phosphorylation sites in L(2)gl are labeled with P. Sro7 has two beta propeller structures (green boxes). The first and the last amino acids are labeled for each of the proteins.

Figure 2. A putative model for the inhibitory activity of Tomosyn under resting conditions and after stimulation

(a) Under resting conditions, a docked, unprimed vesicle forms trans-SNARE complexes via interaction of its membrane-bound VAMP with PM-bound Syntaxin and SNAP-25 to form multiple SNARE complexes. This process renders the vesicle fusion-competent (primed) and it can then fuse upon elevation of [Ca²⁺]_i(b) Tomosyn is recruited through its SNARE motif to areas on the PM enriched with Syntaxin molecules and inhibits SNARE complex formation. (c) After intense stimulation, ROCK phosphorylates Syntaxin at Ser14 and this increases the interaction between Tomosyn and phosphorylated syntaxin, and a greater number of Tomosyn proteins are recruited to the membrane through binding to phosphorylated Syntaxins. (d) Dual inhibition by Tomosyn. In addition to the Syntaxin-Tomosyn interaction, the WD domain enhances oligomerization of cis-SNARE complexes and further reduces vesicle priming. The WD domain may also interact with the cytoskeleton. Thus, under Tomosyn overexpression or upon specific stimulation, Tomosyn can impose greater inhibition by forming complexes with SNAP-25 and Syntaxin or by enhancing oligomerization of cis-SNARE complexes, limiting the amount of uncomplexed PM-bound SNAREs and therefore reducing the formation of trans-SNARE complexes. For clarity, only the SNARE domain of Syntaxin is shown.

Figure 3. Schematic representation of vesicle docking and priming, and Tomosyn's effects on vesicle mobility and turnover

Vesicles undergo morphological docking or tethering through interactions with the subcortical cytoskeleton or with other proteins, such as the exocyst complex or Munc18 (step 1). Following the formation of the first trans-SNARE complex, the vesicle's mobility decreases (step 2). This represents the first step in the priming process and the formation of a functional docked vesicle. Additional SNARE complexes are formed once the vesicle is immobilized, without affecting its mobility (step 3). Upon binding of accessory proteins such as Complexin and/or Synaptotagmin, priming is complete and the vesicle becomes fusion-competent (step 4). Note that the priming process consists of several steps until the vesicle becomes fully fusion-competent. Steps 2 and 3 involve an identical molecular process, the formation of trans-SNARE complexes, although step 2 involves a decline in vesicle mobility and step 3 does not. Tomosyn can interfere with the formation of SNARE complexes in steps 2 or 3. Interfering with step 2 will increase the turnover rate of newly arriving vesicles and inhibit their immobilization. Interfering with step 3 will reduce the fusion-competence of the vesicles and decrease its release probability.