A direct for for SM proteins as templates for SNARE assembly

Abstract

Intracellular membrane trafficking depends on the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that physically drive membrane fusion. Membrane-bridging SNARE complexes are critical for fusion, but their spontaneous assembly occurs inefficiently. To aid in the assembly of fusion competent SNARE complexes, cells rely upon a host of regulatory machinery including Rabs, tethering complexes, and Sec1-Munc18 (SM) family proteins. To elucidate the molecular mechanisms behind this process, we have employed yeast vacuole fusion as a model system and focused our studies on the homotypic fusion and protein sorting (HOPS) tethering complex and the SM protein Vps33, which is a stable member of the HOPS complex.

To better understand the relationship between Vps33 and HOPS, we have determined x-ray structures of Vps33 alone and bound to its HOPS binding partner, Vps16. This interaction is essential for function of the HOPS complex, presumably to localize Vps33 to the site of membrane fusion. As SM proteins are central regulators of SNARE function, we also determined x-ray structures of Vps33 bound to two vacuolar SNAREs, Nyv1 and Vam3. The two SNAREs, one from each membrane, are held in the correct orientation and register for subsequent assembly, suggesting that Vps33 and potentially other SM proteins are templates for generating partially zipped SNARE assembly intermediates. Vps33 mutants that disrupted SNARE binding resolved the tethering and SNARE assembly functions of HOPS and demonstrate that HOPS-mediated SNARE complex assembly is essential at physiologically low SNARE concentrations. Thus, Vps33 catalyzes SNARE complex assembly through specific SNARE motif recognition.