Investigating the conformation of the Qa-SNARE Vam3 and its interactions with the HOPS subcomplex Vps33:Vps16

Abstract

The formation and preservation of functional compartments within metazoan cells both require membrane fusion. Membrane-bridging SNARE complexes are essential catalysts of this process, but without Sec1-Munc18 (SM ) proteins and membrane tethering complexes their assembly is inefficient. The mechanisms through which SNAREs, SM proteins and tethers cooperate for rapid fusion remain enigmatic. A convenient and minimal system to study these interactions comprises the Qa-SNARE Vam3 and two subunits of the HOPS tethering complex: Vps33, an SM protein which templates SNARE assembly, and Vps16, a subunit which recruits Vps33 to HOPS and hence provides access to the tethering function of the complex. Specifically, we focused on the role of the N-terminal domain of Vam3, a non-essential domain which regulates the rate of membrane fusion, in affecting the secondary structure of the protein and its interactions with the Vps33:Vps16 subcomplex. Using circular dichroism spectroscopy, we showed that the N-terminal domain has substantial helical content with 53% of residues involved in α-helical structures. We demonstrate that the N-terminal domain has high thermal stability (Tm = 73 ℃), which exceeds the melting temperature of the cytosolic region of Vam3 by 8 ℃. Additionally, we present data from size-exclusion chromatography which contradict accounts that Vam3-NTD binds to Vps33:Vps16. Taken together, our results suggest that the N-terminal domain interacts with HOPS through other subunits, while the N-terminal domain could be destabilized through an interaction with the SNARE domain of Vam3.