# Autophagosome closure by the ESCRT machinery

> **NIH NIH R01** · PENNSYLVANIA STATE UNIV HERSHEY MED CTR · 2022 · $348,062

## Abstract

Project Summary/Abstract
The goal of this project is to address a fundamental gap in knowledge on how phagophores are closed to form
double membrane autophagosomes. During macroautophagy (hereafter autophagy), crescent-shaped
phagophores elongate around cytoplasmic material and seal to generate double-membrane autophagosomes
that fuse with lysosomes for cargo degradation. As the phagophore rim narrows, the membranes must undergo
fission to separate the inner and outer membranes in a process that bears resemblance to endosomal sorting
complexes required for transport (ESCRT)-mediated membrane scission. Using our elegant HaloTag-LC3
autophagosome completion assay, we provided the first experimental evidence for the ESCRT machinery in
mammalian phagophore closure and identified the ESCRT-I subunit VPS37A as critical factor for the
recruitment of downstream ESCRTs to the phagophore. Notably, we found that the N-terminal putative
ubiquitin E2 variant (PUEV) domain of VPS37A is uniquely required for autophagosome closure but is
dispensable for other ESCRT-mediated membrane abscission processes, including endosome receptor sorting
and cytokinesis. Compartment-specific targeting factors initiate the sequential recruitment of the four ESCRT
complexes (ESCRT-I, -II, -III and VPS4) to the membrane scission site. While the phagophore-specific
targeting factors for the VPS37A-containing ESCRT-I complex are unknown, our preliminary study has
revealed that VPS37A PUEV interacts with highly curved membranes containing anionic lipids and lipid
packing defects, which are all features of the phagophore rim. We hypothesize that the PUEV selectively
interacts with highly curved phagophore membranes to target ESCRT-I to the phagophore. Furthermore, our
preliminary work has revealed that ESCRT recruitment to phagophores requires protein ubiquitylation and the
LC3/GABARAP conjugation machinery, leading us to believe that the stabilization of membrane-associated
ESCRT-I requires additional interactions with phagophore-associated ubiquitylated cargo. We are in an ideal
position to test our hypotheses in the following Specific Aims: (1) to determine how VPS37A targets ESCRT-I
to phagophores and directs the assembly of downstream ESCRTs during autophagosome biogenesis; (2) to
identify phagophore-specific targeting factors for ESCRT-I during autophagy. As autophagy is involved in
numerous physiological and pathological processes, these studies will have far-reaching implications for
human health and disease.

## Key facts

- **NIH application ID:** 10453304
- **Project number:** 2R01GM127954-05
- **Recipient organization:** PENNSYLVANIA STATE UNIV HERSHEY MED CTR
- **Principal Investigator:** HONG-GANG WANG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $348,062
- **Award type:** 2
- **Project period:** 2018-07-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10453304

## Citation

> US National Institutes of Health, RePORTER application 10453304, Autophagosome closure by the ESCRT machinery (2R01GM127954-05). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10453304. Licensed CC0.

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