Biochemical Analysis of a Regulatory Interaction within the HOPS Membrane Tethering Complex

Abstract

Membrane fusion is required for transport of material between organelles and to the extracellular space in eukaryotic cells. SNARE proteins, anchored across membranes, facilitate fusion by zippering into four helix bundles. Although SNAREs are necessary and sufficient for membrane fusion, they function slowly without the aid of other proteins. In vivo, additional protein machineries like Rab GTPases and tethering proteins regulate membrane proximity, while Sec1/Munc18 (SM) proteins catalyze trans-SNARE complex assembly. By functioning both as a tethering factor and SM protein, the HOPS complex regulates all membrane fusion in the endolysosomal pathway. To accomplish these functions, HOPS contains two Rab-binding subunits, which allow it to tether two membranes in space. HOPS’ SM protein subunit, Vps33, then catalyzes SNARE complex assembly by templating SNAREs and fusing the two membranes. The precise mechanism by which HOPS is activated to perform its functions, though, is unknown. Structural biology has been used to gain insight about HOPS’ mechanism of action. Work done by a member of our lab yielded a 4.7 Å resolution cryo-EM structure of HOPS’ SM protein subunit Vps33 in complex with Vps18 and Vps16, two other HOPS subunits. The structure showed surprising evidence of Vps18 binding within an important SNARE binding active site of Vps33, inspiring the hypothesis that Vps18 is an allosteric regulator of Vps33. To characterize the Vps33:Vps18 binding interaction, we sought to use biochemical assays and x-ray crystallography to validate the existing cryo-EM structure and the interactions predicted by it. The data presented in this study suggests that while the cryo-EM structure generally maps Vps18 properly within the Vps33 active site, the specific residue interactions modeled in the structure need further biochemical and structural validation.