An Msp1-Protease Chimera Captures Transient AAA+ Interactions and Unveils Ost4 Mislocalization Errors

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.