Quantitative investigation of the membrane protein degradation mechanism by membrane-integrated AAA protease FtsH under native environments

Summary

"Controlled degradation of misassembled and dispensable proteins is a crucial cellular process for maintaining the quality control of proteomes. In cells, one of the important carriers of this process is AAA+ (ATPases Associated with diverse cellular Activities) proteases, which mediate ATP-dependent proteolysis. The FtsH family proteins are the only membrane integrated AAA+ proteases, which critically contribute to membrane protein degradation. To investigate the mechanisms of membrane... Show more"Controlled degradation of misassembled and dispensable proteins is a crucial cellular process for maintaining the quality control of proteomes. In cells, one of the important carriers of this process is AAA+ (ATPases Associated with diverse cellular Activities) proteases, which mediate ATP-dependent proteolysis. The FtsH family proteins are the only membrane integrated AAA+ proteases, which critically contribute to membrane protein degradation. To investigate the mechanisms of membrane protein degradation mediated by FtsH, I successfully reconstituted the degradation process using FtsH of E. coli in a lipid bilayer environment (Chapter 2). I also developed a six-helical bundle intramembrane protease GlpG of E. coli into a model membrane substrate to study the quantitative relationship between folding and degradation (Chapter 2). I found that FtsH has a substantial ability to accelerate unfolding of membrane substrates up to 800 fold using ATP hydrolysis, and the intrinsic folding properties of the substrates such as local stability, spontaneous unfolding rates, and hydrophobicity also impact degradation rates. Finally, I quantified the total ATP cost that FtsH consumes to degrade membrane proteins (Chapters 3 and 4). To degrade membrane proteins, FtsH needs to overcome large energetic costs for unfolding substrates in the membranes and extracting them towards its protease domain located outside the membrane. I found that FtsH utilizes ATP hydrolysis in degrading membrane proteins with similar efficiency to other AAA+ proteases in degrading water-soluble substrates. This efficiency is achieved by coupling multiple ATP hydrolysis events to degradation in a highly cooperative manner. These findings provide new insights into the physical principles of ATP-dependent degradation of membrane proteins, and the in vitro system developed will serve as a model for further refining the mechanisms of membrane protein degradation."--Pages ii-iii. Show less