Abstract

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.

Highlights

  • Tomosyn regulates vesicle fusion but its mechanism remains incompletely understood

  • The N-terminal domain (NTD) of tomosyn is required for the binding of tomosyn to the syntaxin monomer, indicating that FL tomosyn presents unique features not found in the widely studied C-terminal domain (CTD) fragment. These findings suggest that tomosyn uses its CTD to arrest sensitive factor attachment protein receptors (SNAREs)-dependent membrane fusion, whereas the NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction

  • The NTD of tomosyn, on the other hand, associated with neither the syntaxin-4 monomer nor the syntaxin-41⁄7SNAP-23 complex in the liposome co-flotation assay (Fig. 6E). These results suggest that, whereas the CTD mediates the inhibitory function of tomosyn in SNARE-mediated fusion reaction, both the NTD and CTD are required for the binding of tomosyn to the syntaxin monomer

Read more

Summary

Background

Tomosyn regulates vesicle fusion but its mechanism remains incompletely understood. Results: Tomosyn uses its C-terminal domain to arrest SNARE-dependent fusion reactions, whereas its N-terminal domain is required for syntaxin interaction. The vesicle fusion reaction involves another t-SNARE complex, syntaxin-1 and SNAP-25 [25,26,27] Another widely studied exocytic pathway is the release of neurotransmitters at chemical synapses, which serves the major form of cell-cell communication in the brain [3]. We expressed and purified active FL tomosyn and examined its molecular mechanism of action in reconstituted systems that recapitulate the vesicle fusion reactions of insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The NTD of tomosyn is required ( not sufficient) for the binding of tomosyn to the syntaxin monomer, indicating that FL tomosyn presents unique features not found in the widely studied CTD fragment These findings suggest that tomosyn uses its CTD to arrest SNARE-dependent membrane fusion, whereas the NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction

EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call