# Single-molecule studies of SNARE assembly and lipid transfer

> **NIH NIH R35** · YALE UNIVERSITY · 2024 · $692,354

## Abstract

The overall goal of our research is to gain a greater understanding of the dynamics of
SNARE assembly underlying Ca2+-triggered synaptic vesicle fusion and the driving forces
for directional lipid transport mediated by bridge lipid transfer proteins (LTPs). The
synaptic fusion machinery contains three SNARE proteins (syntaxin-1, SNAP-25, and
VAMP2), Munc18-1, and at least five regulatory proteins (Munc13-1, synaptotagmin-1
(Syt1), complexin, -SNAP, and NSF). The SNAREs couple their ordered folding and
assembly to membrane fusion like a molecular zipper, thereby generating force to bring
the synaptic and plasma membranes into close proximity to induce fusion. Other proteins
chaperone the initial SNARE assembly and then clamp it to form a primed partially
zippered SNARE complex. Ca2+ triggers its further zippering to conclude membrane
fusion. However, the composition and structure of this primed SNARE complex remain
unclear. In the previous funding cycle, we discovered and characterized a tetrameric
complex formed by Munc18-1, Syntaxin-1, VAMP2, and Munc13-1 in solution. Here, we
propose to reconstitute the entire primed complex, which additionally contains complexin
and the Ca2+ sensor Sty1, and characterize its stability, structure, and dynamics in a
membrane environment. As a newly identified category of LTPs in eukaryotes, bridge
LTPs are thought to act as a conduit to enable lipids to flow from one membrane to the
other at their contact site. The bulk lipid flow causes membrane expansion of certain
target membranes, leading to organelle biogenesis. However, the forces that dictate the
direction and rate of the lipid flow are unknown. We propose that gradients of membrane
tension and membrane protein densities can serve as the long-thought driving forces,
which will be tested in our novel lipid transfer assays. SNARE-mediated vesicle fusion
and LTP-mediate lipid transfer are two complementary membrane trafficking pathways,
whose malfunction has been linked to numerous diseases. A better understanding of their
mechanisms has the potential to enable intervention in these diseases.

## Key facts

- **NIH application ID:** 10765310
- **Project number:** 2R35GM131714-06
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Yongli Zhang
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $692,354
- **Award type:** 2
- **Project period:** 2019-05-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10765310

## Citation

> US National Institutes of Health, RePORTER application 10765310, Single-molecule studies of SNARE assembly and lipid transfer (2R35GM131714-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10765310. Licensed CC0.

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