Energy dependent proteolysis is essential for all life, but uncontrolled degradation leads to devastating consequences. In bacteria, oligomeric AAA+ proteases are responsible for controlling protein destruction and are regulated in part by adaptor proteins. Adaptors are regulatory factors that shape protease substrate choice by either restricting or enhancing substrate recognition in several ways. In some cases, protease activity or assembly itself requires adaptor binding. Adaptors can also alter specificity by acting as scaffolds to tether particular substrates to already active proteases. Finally, hierarchical assembly of adaptors can use combinations of several activities to enhance the protease’s selectivity. Because the lifetime of the constituent proteins directly affects the duration of a particular signaling pathway, regulated proteolysis impacts almost all cellular responses. In this review, we describe recent progress in regulated protein degradation, focusing on fundamental principles of adaptors and how they perform critical biological functions, such as promoting cell cycle progression and quality control.
Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In Caulobacter crescentus, cell cycle–dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.
25Bacterial protein degradation is a regulated process aided by protease adaptors that alter 26 specificity of energy dependent proteases. In Caulobacter crescentus, cell-cycle 27 dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric 28 RcdA adaptor which binds multiple cargo and delivers substrates to the ClpXP protease. 29RcdA itself is degraded in the absence of cargo and how RcdA recognizes its targets is 30 unknown. Here we show that RcdA dimerization and cargo binding compete for a 31 common interface. Cargo binding separates RcdA dimers and a monomeric variant of 32 RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. 33Based on HDX-MS studies showing that different cargo rely on different regions of the 34 dimerization interface, we generate RcdA variants that are selective for specific cargo and 35 show cellular defects consistent with changes in selectivity. Using the same interface for 36 dimerization and cargo binding offers an ability to limit excess protease adaptors by self-37 degradation, while providing capacity for binding a range of substrates. 38 39 40 Significance Statement: 41 Energy-dependent proteases broadly regulate bacterial physiology and development. 42 Adaptor proteins tune the substrate specificity of proteases to only degrade selective 43 substrates during the bacterial life cycle and during times of cellular stress. In the 44 process of delivering cargo to their respective proteases, adaptor proteins are inherently 45 protected from degradation until the delivery is complete. How protease adaptors can 46 recognize a wide range of cargo while maintaining stringent specificity and how this 47 process results in stabilization of adaptors remains unclear. Here, we show that direct 48 competition for distinct regions of the dimer interface of the RcdA adaptor by its cargo 49 protects RcdA from degradation by the ClpXP protease, and that this interface can be 50 selectively perturbed in a rational manner with biochemical and physiological 51 consequences. 52 53 Highlights: 54 Cargo binding of RcdA cargo competes with dimerization 55 Dimerization of RcdA is necessary for self-degradation by ClpXP 56 RcdA can deliver either cargo or other RcdA subunits to ClpXP 57 Different regions of the dimerization interface are needed for different cargo 58 59
Bacterial protein degradation is a tightly coordinated process aided by protease adaptors that coordinate cell cycle and manages stress responses. In Caulobacter crescentus, cell‐cycle dependent protein degradation depends on the highly conserved ClpXP protease and several ClpXP specific adaptors that dictate hierarchal substrate degradation. While most protease adaptors deliver only a single substrate, the RcdA adaptor directly binds and coordinates the degradation of several cell‐cycle regulated substrates that share no obvious sequence or structural similarities. Here we show that cargo binding uses the same interface as that responsible for dimerization of RcdA. Cargo binding converts RcdA from a homodimer to a monomer and this effect extends to all RcdA cargo. We identify key residues in this interface with hydrogen‐deuterium exchange mass spectrometry and make mutations to generate a monomeric variant incapable of dimerization that also fails to bind cargo. Interestingly, RcdA itself is normally degraded by ClpXP unless bound to cargo, but the monomeric variant fails to be degraded, suggesting that RcdA delivers cargo or delivers itself to ClpXP. Expressing a monomeric RcdA in vivo results in irregular stalk formation and loss of normal cargo turnover. Our work shows how cargo binding competes with self‐dimerization of RcdA through direct competition for a common interface and that disruption of this interface results in defects in normal cell physiology.
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