A Biochemical Analysis of the Interaction Between the Sec1/Munc18 (SM) Protein Vps45 and the Qa-SNARE Tlg2

Abstract

N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) assemble into a four-helix bundle that promotes membrane fusion in vesicle trafficking. The formation of a SNARE bundle is regulated by a number of chaperones, including members of the Sec1/Munc18 (SM) protein family. In particular, SM proteins interact with Qa-SNAREs in various ways that suggest positive and negative roles of SM proteins on SNARE complex assembly. A conserved mechanism by which SM proteins bind to Qa-SNAREs to mediate SNARE bundle formation is an intriguing possibility that deserves further investigation. The endosomal SM protein Vps45 interacts with the Qa-SNARE Tlg2 by the binding modes reported for other SM proteins and their cognate SNAREs. Characterizing the interaction between Vps45 and Tlg2 has the potential to provide new insights into SM protein function. Here we present a biochemical analysis of the binding between Chaetomium thermophilum Vps45 and Tlg2. Binding assays reveal that Vps45 binds to multimeric and monomeric forms of Tlg2, an interaction mediated by the Tlg2 N-peptide. Vps45 did not promote SNARE bundle assembly under the conditions we tested. Instead, our findings suggest a model in which Vps45 protects Tlg2 from self-association and in combination with other factors, enables monomeric Tlg2 SNARE to be incorporated into a SNARE bundle. Future work to investigate the multimerization of Tlg2 and to pursue a structural model of Vps45 binding Tlg2 will further the understanding of how SM proteins interact with Qa-SNAREs for SNARE complex assembly.