# Mechanisms of membrane tethering in autophagy

> **NIH NIH R35** · UNIVERSITY OF MISSOURI-COLUMBIA · 2024 · $381,212

## Abstract

Project Summary/Abstract
Cells are in a constant battle to maintain homeostasis and respond to stress. Autophagy is a conserved
eukaryotic pathway that responds to cellular stresses. Autophagy identifies and encapsulates cellular debris
in an autophagosome, which is ultimately fused with the lysosome for degradation. Studies have shown that
the final step of autophagy, termed autolysosomal fusion, requires several factors. This includes membrane
effector proteins, Rabs and Atg8 homologs, multifunctional scaffolding proteins and specialized lipid
headgroups (e.g. PI3P). The assembly of proteins ‘hubs’ often promote cellular processes and are required
for function. The Homotypic fusion and vacuole Protein Sorting (HOPS) complex, Ectopic P-Granules 5
Autophagy Tethering Factor (EPG-5), and Pleckstrin homology domain-containing family M member 1
(PLEKHM1) are scaffolding proteins present at the final steps of autophagy. Mutations in these multifunctional
scaffolds lead to poor cellular health and have been implicated in several human diseases, specifically defects
in these proteins lead to neurodegenerative diseases. Despite the importance of the final stages of autophagy,
we currently lack fundamental information on how these interaction hubs tether and organize sites of
autolysosomal fusion.
The overarching goal of this proposal is to resolve the protein interaction network that drive autolysosomal
tethering. We are initiating both in vitro and in vivo techniques to discovery the molecular interactions that
drive autolysosomal tethering. Single particle cryo-electron microscopy analysis will serve as our main tool to
determine how the human HOPS complex engages with autophagy adaptor PLEKHM1 at the membrane
surface. These studies have the potential to reveal the molecular interactions which drive the formation of the
autolysosomal interface and generate specificity within the autophagy pathway. In tandem, we will utilize cryo-
focused ion beam (cryo-FIB) milling along with in situ cryo-electron tomography methods to examine
autolysosomal tethering in the native cellular environment. To accomplish this, we will focus on EPG-5, a
scaffold that binds to both lysosome and autophagosome directly (via protein-protein interactions with Rab7
and Atg8 homologs, respectively). EPG-5 is an ideal target for in situ studies given its large size (300 kDa)
and distinct shape. By using cell biological techniques, we will enrich autolysosomal tethering events and
perform cryo-FIB milling. These innovative approaches have the potential to discover the cellular context of
autolysosomal fusion at resolutions (<20Å) not possible by traditional techniques. Taken together, our work
will provide to a deeper understanding at both an atomic level and contextual level. Long term, we hope our
data contributes to novel therapeutic approaches to treat membrane tethering defects to improve human
health. Moreover, the principles we discover could lay a foundation for understandin...

## Key facts

- **NIH application ID:** 10940097
- **Project number:** 1R35GM155253-01
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** Adam Lee Yokom
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $381,212
- **Award type:** 1
- **Project period:** 2024-07-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10940097

## Citation

> US National Institutes of Health, RePORTER application 10940097, Mechanisms of membrane tethering in autophagy (1R35GM155253-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10940097. Licensed CC0.

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