Biochemical Studies of SNARE Regulation: Interaction of the HOPS Subunit Vps18 with the Vacuolar SNARE Vam7

Abstract

The HOPS complex plays an indispensible role in early endosome, late endosome and lysosome formation. In addition to its tethering function, HOPS is also implicated in regulating SNARE complex assembly. However, currently there is no structural mechanism for this process. The HOPS subunits Vps33 and Vps16 are under intense scrutiny due to their interactions with the SNARE complex. However, a third HOPS subunit, Vps18, has been reported to interact with the soluble SNARE Vam7. HOPS recruitment of Vam7 facilitates SNARE complex formation and increases the efficiency of membrane fusion in vitro. Here we use size-exclusion binding assays using various constructs of Vam7 and Vps18 from Chaetomium thermophilum to better define the binding interface between Vps18 and Vam7. These data combined with previous genetic and biochemical experiments can provide a more detailed model of HOPS-mediated SNARE regulation.