# Structural Analysis of Membrane Tethering and Fusion Proteins

> **NIH NIH R01** · PRINCETON UNIVERSITY · 2020 · $359,027

## Abstract

PROJECT SUMMARY / ABSTRACT
Most membrane fusion reactions in eukaryotic cells are executed by membrane-bridging SNARE complexes.
The formation of these complexes generally 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. Consequently, we hypothesize that most SNARE complex assembly reactions in the cell
are orchestrated by a set of `topologically aware' chaperones called multisubunit tethering complexes (MTCs).
These highly-conserved nanomachines interact directly with virtually all of the proteins (including the SNAREs)
implicated in membrane tethering and fusion. 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 in the assembly of fusogenic SNARE complexes. In
Aim 1, we will conduct structural studies of the homotypic fusion and vacuole protein sorting (HOPS) complex,
a well-studied MTC required for fusion at late endosomes and lysosomes/vacuoles. High-resolution cryo-
electron microscopy will be used to elucidate the structure of the HOPS complex. In order to elucidate how
HOPS organizes SNAREs for assembly, we will also determine crystal structures of complexes between
HOPS subunits and SNARE N-terminal regulatory domains. These structures will then serve as blueprints for
in vivo and in vitro functional studies. In Aim 2, we will elucidate the detailed mechanism by which a subunit of
the HOPS complex, the SM protein Vps33, catalyzes SNARE assembly. These studies will encompass
fluorescence-based biochemical assays, crystal structures of SNARE assembly intermediates, reconstituted
proteoliposome-based fusion assays, and single-molecule optical tweezers experiments. In Aim 3, we will
expand these studies to other SM proteins, using the same suite of experimental approaches to test the
generality of our Vps33-derived mechanistic model. These experiments will also afford us an opportunity to
evaluate the extent to which the catalytic activity of SM proteins is under regulatory control. Finally, in Aim 4,
we will turn our attention to the simplest known MTC, the Dsl1 complex. Using X-ray crystallography and in
vivo imaging, we will rigorously test the role of the Dsl1 complex in vesicle tethering and tethering-triggered
vesicle uncoating.

## Key facts

- **NIH application ID:** 9878872
- **Project number:** 5R01GM071574-16
- **Recipient organization:** PRINCETON UNIVERSITY
- **Principal Investigator:** FREDERICK M HUGHSON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $359,027
- **Award type:** 5
- **Project period:** 2005-03-01 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9878872

## Citation

> US National Institutes of Health, RePORTER application 9878872, Structural Analysis of Membrane Tethering and Fusion Proteins (5R01GM071574-16). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9878872. Licensed CC0.

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