Abstract
ClpX is a member of the Clp/Hsp100 family of ATP-dependent chaperones and partners with ClpP, a compartmentalized protease, to degrade protein substrates bearing specific recognition signals. ClpX targets specific proteins for degradation directly or with substrate-specific adaptor proteins. Native substrates of ClpXP include proteins that form large oligomeric assemblies, such as MuA, FtsZ, and Dps in Escherichia coli. To remodel large oligomeric substrates, ClpX utilizes multivalent targeting strategies and discriminates between assembled and unassembled substrate conformations. Although ClpX and ClpP are known to associate with protein aggregates in E. coli, a potential role for ClpXP in disaggregation remains poorly characterized. Here, we discuss strategies utilized by ClpX to recognize native and non-native protein aggregates and the mechanisms by which ClpX alone, and with ClpP, remodels the conformations of various aggregates. We show that ClpX promotes the disassembly and reactivation of aggregated Gfp-ssrA through specific substrate remodeling. In the presence of ClpP, ClpX promotes disassembly and degradation of aggregated substrates bearing specific ClpX recognition signals, including heat-aggregated Gfp-ssrA, as well as polymeric and heat-aggregated FtsZ, which is a native ClpXP substrate in E. coli. Finally, we show that ClpX is present in insoluble aggregates and prevents the accumulation of thermal FtsZ aggregates in vivo, suggesting that ClpXP participates in the management of aggregates bearing ClpX recognition signals.
Highlights
Maintaining cellular proteostasis relies on chaperone pathways that promote native protein folding
The heat inactivation is irreversible since incubation of aggregated Gfp-ssrA alone does not lead to appreciable fluorescence reactivation, which is consistent with previous reports using Gfp (Figure S1; Zietkiewicz et al, 2004)
We observed that ClpXP incubated at 30◦C was more active than ClpXP incubated at 50◦C, suggesting that a partial loss of activity had occurred at high temperature (Figure S4F). This assay was performed in the complete absence of other cellular chaperones or substrates and suggests that some ClpXP likely continues to retain activity after exposure to heat stress, while some may become inactivated. Using both a native and an engineered aggregated substrate, we demonstrate that ClpXP has the operational capacity to disassemble and degrade large aggregates that have ClpX degrons
Summary
Maintaining cellular proteostasis relies on chaperone pathways that promote native protein folding. Typical strategies include targeting misfolded, unfolded, and aggregated polypeptides for reactivation or degradation (Bukau and Horwich, 1998; Wickner et al, 1999; Stoecklin and Bukau, 2013). Misfolded proteins are generated during polypeptide elongation and as a complication of environmental stress (Powers and Balch, 2013). The challenges imposed on chaperone systems by proteotoxic stress are especially relevant in pathogenic organisms like E. coli, which experience. ClpXP Targets Aggregates for Disassembly extreme fluctuations in environmental conditions leading to accumulation of protein aggregates and subsequent proteotoxicity (Mogk et al, 2011). Under thermal stress in E. coli, the heat shock response provides a cellular defense mechanism and upregulates heat shock protein and chaperone levels to restore proteostasis (Mogk et al, 2011)
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