# ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA BERKELEY · 2020 · $286,298

## Abstract

Project Summary
Protein degradation in all prokaryotic and eukaryotic cells is tightly regulated by ATP-dependent
compartmental proteases. These enzymes of the AAA+ family use ATP hydrolysis in a hexameric ATPase
motor to drive the mechanical unfolding of protein substrates and their translocation into the sequestered
chamber of an associated peptidase for degradation. The major ATP-dependent protease in eukaryotic cells is
the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and
thereby controls protein homeostasis as well as numerous vital processes, including transcription, cell division,
differentiation, signal transduction, and apoptosis. Despite the great importance of the 26S proteasome for cell
viability, its detailed mechanisms for substrate processing and regulation still remain largely elusive. Over the
past five years, we were already able to significantly advance our understanding of proteasome structure and
function. We established heterologous E. coli-expression systems for the yeast proteasome lid and base
subcomplexes, which together with the in-vitro reconstitution of partially recombinant 26S holoenzymes
revolutionized mutational, mechanistic, and structural studies of the proteasome. Using cryo-EM, we revealed
the complete subunit architecture of the proteasome and discovered major substrate-induced conformational
changes that allow deubiquitination, unfolding, and processive translocation. Furthermore, we were able to
uncover that the individual ATPase subunits differentially contribute to the activities of the heterohexameric
AAA+ motor, and we provided novel biophysical insights into forceful protein unfolding and the
mechanochemistry of ATP-dependent proteases by performing single-molecule optical-tweezers
measurements on the related bacterial protease ClpXP. Our established biochemical tools, recombinant
systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous
outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S
proteasome and other AAA+ motors, as well as the regulation of pathways connected to the ubiquitin-
proteasome system. We will employ a multidisciplinary approach that includes in-vitro biochemical, single-
molecule, and atomic-resolution structural studies. Our mechanistic dissection of proteasome function and
regulation, together with the characterization of important determinants for cellular substrate selection, will
open numerous future opportunities for extending our research to other crucial pathways that feed into or are
regulated by the ubiquitin-proteasome system. We are expanding our research to the AAA+ translocase
Pex1/Pex6, which is essential for peroxisome biogenesis and delivers ubiquitinated proteins to the proteasome
for degradation. Due to the important regulatory functions of the proteasome and its role in cancer biology, our
research also has subs...

## Key facts

- **NIH application ID:** 9932405
- **Project number:** 5R01GM094497-09
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Andreas Martin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $286,298
- **Award type:** 5
- **Project period:** 2011-07-01 → 2021-08-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9932405

## Citation

> US National Institutes of Health, RePORTER application 9932405, ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome (5R01GM094497-09). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9932405. Licensed CC0.

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