Related Experiment Video
Updated: May 13, 2025
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
An Msp1-Protease Chimera Captures Transient AAA+ Interactions and Unveils Ost4 Mislocalization Errors
Abstract:
Membrane protein homeostasis (proteostasis) is essential for maintaining the integrity of eukaryotic organelles. Msp1 is a membrane anchored AAA+ (ATPase Associated with cellular Activities) protein that maintains mitochondrial proteostasis by extracting aberrant proteins from the outer mitochondrial membrane. A comprehensive understanding of the physiological roles of Msp1 has been hindered because AAA+ proteins interact with substrates transiently and common strategies to stabilize this interaction lead to undesirable mitochondrial phenotypes. To circumvent these drawbacks, we fused catalytically active Msp1 to the inactivated protease domain of the AAA+ protease Yme1. The resulting chimera sequesters substrates in the catalytically inactive degradation chamber formed by the protease domain. We performed mass spectrometry analysis with the Msp1-protease chimera and identified the signal anchored protein Ost4 as a novel Msp1 substrate. Topology experiments show that Ost4 adopts mixed orientations when mislocalized to mitochondria and that Msp1 extracts mislocalized Ost4 regardless of orientation. Together, this work develops new tools for capturing transient interactions with AAA+ proteins, identifies new Msp1 substrates, and shows a surprising error in targeting of Ost4.
Insights
Msp1 protein homeostasis is vital for mitochondria. Researchers developed a new chimera tool to identify Msp1 substrates, discovering Ost4 as a novel target and revealing errors in its mitochondrial targeting.
Area of Science:
- Cell Biology
- Molecular Biology
- Mitochondrial Biology
Background:
- Membrane protein homeostasis (proteostasis) is crucial for eukaryotic organelle integrity.
- Msp1, a membrane-anchored AAA+ ATPase, maintains mitochondrial proteostasis by extracting aberrant proteins.
- Studying transient AAA+ protein-substrate interactions is challenging due to stabilization strategies causing mitochondrial issues.
Purpose of the Study:
- To develop novel tools for capturing transient interactions of AAA+ proteins.
- To identify novel substrates of the mitochondrial AAA+ protein Msp1.
- To investigate the targeting and extraction mechanisms of Msp1 substrates.
Main Methods:
- Construction of a Msp1-protease chimera to stabilize substrate interactions.
- Mass spectrometry analysis using the chimera to identify Msp1 substrates.
- Topology experiments to analyze substrate orientation and Msp1 extraction efficiency.
Main Results:
- The Msp1-protease chimera successfully sequestered substrates for analysis.
- The signal-anchored protein Ost4 was identified as a novel Msp1 substrate.
- Msp1 extracts mislocalized Ost4 from mitochondria irrespective of its orientation.
Conclusions:
- The Msp1-protease chimera is an effective tool for studying AAA+ protein interactions.
- Ost4 is a newly identified Msp1 substrate, highlighting a role for Msp1 in its mitochondrial quality control.
- Mispreading of Ost4 to mitochondria occurs with mixed orientations, and Msp1 corrects this error.
