# Defining the molecular mechanisms underlying mitochondrial proteostasis

> **NIH NIH R21** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $290,250

## Abstract

Project Summary
A critical factor in defining mitochondrial dysfunction during normal aging and age-associated disease is the
maintenance of mitochondrial protein integrity (also referred to as protein homeostasis or proteostasis).
Mitochondrial proteostasis is primarily regulated by ATP-dependent quality control proteases, including the
soluble proteases, LON and CLPXP, and the membrane associated proteases, YME1L and AFG3L2. These
quality control proteases regulate all aspects of mitochondrial function including energy metabolism, lipid
synthesis, and apoptotic signaling. Mitochondrial quality control proteases adopt a similar “stacked ring”
organization, wherein evolutionarily similar AAA+ ATPase domains undergo ATP-dependent rearrangements
to translocate protein substrates through a central pore to a proteolytic chamber for cleavage. Despite the
evident structural and mechanistic similarities, mitochondrial proteases exhibit distinct proteolytic activities that
allow them to regulate specific mitochondrial function. A consequence of the distinct functions of these
proteases is that genetic or age-related alterations in the activity of specific proteases distinctly influences
mitochondrial and cellular physiology in the context of organismal aging. Thus, an important question is “How
do these mitochondrial quality control proteases use a similar architecture to differentially influence specific
aspects of mitochondrial biology?” Here, we hypothesize that unique structural features in each of these
proteases have been incorporated into a generally conserved mechanism of ATP-dependent protease
activity, endowing mitochondrial quality control proteases the capacity for unique biologic function.
We solved the first near atomic resolution cryo-electron microscopy (cryo-EM) structure of the catalytic core of
the yeast YME1L homolog YME1 bound to nucleotides and a peptide substrate, revealing the molecular
mechanism responsible for ATP-dependent substrate engagement and translocation into the proteolytic
chamber. We are now defining similar substrate-bound structures for the soluble AAA+ proteases LON and
CLPXP using an analogous cryo-EM approach. Furthermore, we are establishing a novel unnatural amino acid
platform to isolate the full-length membrane integrated (IM) proteases YME1L and AFG3L2 for structure
determination by cryo-EM. By comparing structures of these evolutionarily related proteases, we are identifying
the shared mechanisms that drive substrate translocation, as well as the unique structural features critical for
their specific protease activities. These differences will reveal the molecular mechanisms by which aging or
mutation may influence the activity of proteases, and identify new opportunities to therapeutically influence
mitochondrial proteolytic activity to prevent aging- or disease-associated mitochondrial dysfunction by targeting
specific aspects of protease structure.

## Key facts

- **NIH application ID:** 9852940
- **Project number:** 5R21AG061697-02
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Gabriel C Lander
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $290,250
- **Award type:** 5
- **Project period:** 2019-02-01 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9852940

## Citation

> US National Institutes of Health, RePORTER application 9852940, Defining the molecular mechanisms underlying mitochondrial proteostasis (5R21AG061697-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9852940. Licensed CC0.

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