Exosomes play crucial roles in local and distant cellular communication and are involved in various physiological and pathological processes. Tumour‐derived exosomes are pivotal to tumorigenesis, but the precise mechanisms underlying their secretion remain elusive. In particular, the SNARE proteins that mediate the fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in tumour cells are subject to debate. In this study, we identified syntaxin‐4, SNAP‐23, and VAMP‐7 as the SNAREs responsible for exosome secretion in MCF‐7 breast cancer cells and found that a SNARE complex consisting of these SNAREs can drive membrane fusion in vitro. Deletion of any of these SNAREs in MCF‐7 cells did not affect MVB biogenesis and transportation, indicating their specific involvement in MVB–PM fusion. In addition, syntaxin‐4, SNAP‐23, and VAMP‐7 play equivalent roles in exosome secretion in both HeLa cervical cancer cells and A375 melanoma cells, suggesting their conserved function in exosome secretion. Furthermore, deletion of VAMP‐7 in 4T1 mammary carcinoma cells efficiently inhibited exosome secretion and led to significant attenuation of tumour growth and lung metastasis in mouse models, implying that VAMP‐7 may hold promise as a novel therapeutic target for breast cancer.

The transition of the Munc18-1/syntaxin-1 complex to the SNARE complex, a key step involved in exocytosis, is regulated by Munc13-1, SNAP-25 and synaptobrevin-2, but the underlying mechanism remains elusive. Here, we identify an interaction between Munc13-1 and the membrane-proximal linker region of synaptobrevin-2, and reveal its essential role in transition and exocytosis. Upon this interaction, Munc13-1 not only recruits synaptobrevin-2-embedded vesicles to the target membrane but also renders the synaptobrevin-2 SNARE motif more accessible to the Munc18-1/syntaxin-1 complex. Afterward, the entry of SNAP-25 leads to a half-zippered SNARE assembly, which eventually dissociates the Munc18-1/syntaxin-1 complex to complete SNARE complex formation. Our data suggest that Munc18-1 and Munc13-1 together serve as a functional template to orchestrate SNARE complex assembly.

Synaptotagmin-1 (Syt1) acts as a Ca²⁺ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca²⁺-binding loops, the side polybasic patch, and the bottom face in response to Ca²⁺. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo.