# Mechanistic Investigation of Proteostasis at the Outer Mitochondrial Membrane

> **NIH NIH R35** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2022 · $51,729

## Abstract

Mechanism of Msp1 Mediated Protein Extraction from the Mitochondrial Membrane
As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human
health. Proper mitochondrial function depends on a robust quality control system to maintain homeostasis of
the proteome (proteostasis). Declines in mitochondrial function and/or proteostasis have been linked to cancer,
aging, cardiovascular, and neurodegenerative diseases. A poorly understood aspect of mitochondrial
proteostasis is the removal of membrane proteins from the lipid bilayer. This is due to the technical challenges
of reconstituting this process in vitro. We have overcome this technical barrier by developing a simple, but
powerful reconstituted system with the AAA+ (ATPase Associated with cellular Activities) protein Msp1.
Anchored in the outer mitochondrial membrane (OMM), Msp1 maintains mitochondrial proteostasis by
removing unwanted proteins from the lipid bilayer. Mutations in Msp1 or the human homologue ATAD1 lead to
compromised mitochondrial function, impaired fear conditioning, severe encephalopathy, and early death.
Despite its clear physiological importance, there are many important unanswered questions regarding Msp1
activity. How does Msp1 interact with other quality control components to maintain membrane proteostasis?
What is the full range of substrates extracted by Msp1/ATAD1? How is this regulated? These are particularly
pressing questions given that our collaborator, Jared Rutter (HHMI, University of Utah), has preliminary genetic
evidence that ATAD1 may regulate apoptosis by extracting BH3-only proteins from the OMM. We will use an
unbiased proteomic approach and our in vitro extraction assay to directly test this paradigm shifting hypothesis
and examine the molecular details for how this process is regulated. Because our reconstituted system
overcomes key technical barriers that have hampered previous attempts to study the extraction of membrane
proteins from a lipid bilayer, we will also utilize our system to draw foundational conclusions about how key
factors such as membrane fluidity, substrate structure, and ATP hydrolysis rates affect this essential cellular
process. This work will test an exciting new hypothesis for apoptotic regulation, provide a comprehensive
picture of Msp1/ATAD1 function in mitochondrial biology, and uncover new insights into the fundamental
process of membrane protein extraction.

## Key facts

- **NIH application ID:** 10873385
- **Project number:** 7R35GM137904-04
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Matthew Lee Wohlever
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $51,729
- **Award type:** 7
- **Project period:** 2023-08-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10873385

## Citation

> US National Institutes of Health, RePORTER application 10873385, Mechanistic Investigation of Proteostasis at the Outer Mitochondrial Membrane (7R35GM137904-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10873385. Licensed CC0.

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