ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

Prakash Koodathingal,Neil E Jaffe,Daniel A Kraut,Sumit Prakash,Susan Fishbain,Christophe Herman,Andreas Matouschek

doi:10.1074/jbc.m900783200

Prakash Koodathingal, Neil E Jaffe + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m900783200

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Abstract

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.

Highlights

Energy-dependent proteolysis is responsible for more than 90% of the protein turnover inside the cell [1]
In Archaea, analogous functions are performed by the archaebacterial proteasome, consisting of the proteasome-activating nucleotidase (PAN),3 working with the 20 S proteasome [4]; in the cytoplasm and nucleus of eukaryotes, these same functions are performed by the 26 S proteasome [5]
Regulatory particles belonging to the AAA family of molecular chaperones assemble on either end of the proteolytic chamber and recognize substrates destined for degradation

Summary

EXPERIMENTAL PROCEDURES

Substrates—Protease substrates contained domains derived from Bacillus amyloliquefaciens barnase [31], B. amyloliquefaciens barstar [32], E. coli DHFR [33], E. coli chemotaxis response regulator CheY [34], or mouse DHFR [35]. Fractions containing ClpX were dialyzed, applied to a Q-Sepharose column (GE Healthcare), and eluted with a 0.1–1.0 M KCl gradient in buffer QS (50 mM Tris-HCl, pH 8, 5 mM MgCl2, 5 mM DTT, 10% glycerol). Fractions containing ClpX were loaded onto a Mono Q column (GE Healthcare) and eluted with a 0.1– 0.5 M KCl gradient in buffer MQ (50 mM Tris-HCl, pH 8, 5 mM MgCl2, 5 mM DTT, 10% glycerol). Soluble lysate was applied to a Q-Sepharose column (GE Healthcare) in 10 mM Na2PO4, pH 7.0, 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 10% glycerol and eluted with a 0.1 to 0.4 M KCl gradient Eluates containing both HslU and HslV were pooled and precipitated in 70% saturated (NH4)2SO4. Reactions (50 ␮l) were performed in triplicate at 30 °C

RESULTS

26 S proteasome has a strong unfolding ability but a moderate

DISCUSSION

44 Ϯ 5 72 Ϯ 8