Structural Analysis of Membrane Tethering and Fusion Proteins

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PROJECT SUMMARY / ABSTRACT The traffic patterns established by transport vesicles are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Cargo transported by these vesicles is delivered through the fusion of the vesicle with the membrane of a target organelle or, in the case of exocytosis, the plasma membrane. Membrane fusion is executed by SNARE complexes that bridge the vesicle and target membranes. The formation of these complexes requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. We hypothesize that SNARE complex assembly reactions in the cell are orchestrated by `topologically aware' chaperones called multisubunit tethering complexes (MTCs). We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with—and sometimes as integral subunits of—the MTCs. Therefore, the overarching goal of this proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function, especially as they relate to one another, in the assembly of membrane fusogenic SNARE complexes. Aim 1 is focused on SM proteins with the goal of characterizing their precise catalytic role in SNARE complex assembly. Principally through the use of X-ray crystallography and complementary single-molecule optical tweezers experiments, we will determine the structures and thermodynamic stabilities of SM-bound SNARE assembly intermediates. In Aims 2 and 3, we broaden our focus to include MTCs. In Aim 2, we will investigate the simplest known MTC, the yeast Dsl1 complex, and its interactions with SNAREs and the SM protein Sly1. Cryo-EM studies of arrested SNARE assembly intermediates in complex with both the Dsl1 complex and Sly1 are designed to reveal how the Dsl1 complex and Sly1 collaborate. In Aim 3, we will turn our attention to the homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC that is required for fusion at late endosomes and lysosomes/vacuoles. Importantly, HOPS contains an SM protein as an integral subunit, making it an ideal system for studying MTC–SM collaboration. In order to elucidate how HOPS organizes SNAREs for assembly, we will expand our ongoing cryo-EM studies of HOPS to include bound SNAREs and SNARE assembly intermediates. Overall, this research program has the potential to revolutionize our mechanistic understanding of chaperoned SNARE complex assembly, with potentially profound implications for elucidating diverse biological processes and their subversion during infection and disease. While the proposed work is more fundamental than applied, it will lay a foundation for efforts to manipulate trafficking and other processes entailing membrane fusion, with potential future applications to therapeutic intervention.