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Updated: Jun 8, 2025

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation
Published on: November 6, 2014
Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells
1Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan; Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; Department of Pathology and Cell Biology, Columbia University, New York, NY, United States.
Abstract:
Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to (1) study the turnover and ubiquitination of candidate MAD substrates, (2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and (3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.
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