Background: Qabc-and R-SNARE proteins are key components for membrane fusion in eukaryotic organelles. Results: Reconstituted SNARE proteoliposomal study reveals distinct contributions of Qabc-and R-SNAREs to the specificity of membrane fusion. Conclusion: An assembly of proper Qabc-SNARE combinations is critical for mediating fusion specificity. Significance: This provides new insights for understanding how cells organize their complex membrane trafficking pathways.