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In vivo and in vitro Studies of Adaptor-clathrin Interaction
Published on: January 26, 2011
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Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function
Xiao Wang1, Sanford M Simon1, Philip Coffino1
1Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York, USA.
The Journal of Biological Chemistry
|September 5, 2022
Summary
Substrate tail length and sequence influence how bacterial AAA+ (ATPases Associated with diverse cellular Activities) proteases like ClpX retain or release proteins during unfolding. Specific motifs can inhibit unfolding and affect degradation rates.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Degradation
Background:
- AAA+ proteases unfold proteins via a narrow pore, requiring substrate association.
- Substrate sequence and tail characteristics are known to influence unfolding efficiency.
- Understanding substrate interactions is crucial for AAA+ protease function.
Purpose of the Study:
- To investigate how substrate tail length and composition affect dissociation from the ClpX unfoldase.
- To determine the impact of specific substrate motifs on unfolding, degradation, and ATPase activity.
- To elucidate the relationship between substrate domain stability and tail sequence effects.
Main Methods:
- Single-molecule dwell time measurements using total internal reflection fluorescence microscopy.
- Utilized substrates with stable domains and C-terminal tails of varying lengths and compositions.
- Assessed substrate degradation rates and ATP consumption using the ClpXP unfoldase-protease complex.
Main Results:
- Increased tail length enhanced substrate retention during unfolding attempts.
- Poly-glycine tracts promoted substrate release near the folded domain, while glycine-alanine repeats did not.
- High complexity motifs also promoted substrate release.
- Substrate domain stability modulated the effects of tail sequences on degradation and ATPase activity.
Conclusions:
- Substrate tail characteristics play a significant role in regulating AAA+ protease engagement and unfolding.
- Specific sequence motifs differentially impact substrate release and degradation.
- The interplay between substrate domain stability and tail sequence dictates overall processing by ClpXP.
