The expanded specificity and physiological role of a widespread N-degron recognin

Abstract

All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacteriumSalmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

“…2e). These included mutants mapping to the relA ppGpp synthetase and to an ortholog of the bpt leucine aminoacyl protein transferase involved in N-end-rule degradation 52,53 (r = 0.72-0.87, p < 10 −5 , Fig. 2f).…”

Progression from remodeling to hibernation of ribosomes in zinc-starved mycobacteria

“…The N-terminal sequence of Mrf, however, remained unchanged in high- and low-zinc medium ( SI Appendix , Fig. S6 A – D ), indicating that zinc-dependent degron formation in Mrf likely follows a noncanonical pathway, such as those described in Salmonella enterica ( 36 ). Because Mrf has the signature zinc-binding C 64 XXC 67 motif, which is flanked by four His (H 36,41,62,81 ) ( Fig.…”

“…S5 A – D ). Mycobacterial ClpP1P2 subunits are known to form a barrel-shaped hetero-tetradecameric peptidase complex ( 29 ), representing AAA+ family proteolytic machines that partner with an ATPase (ClpA/ClpC or ClpX) and an adaptor protein (SspB or ClpS) to degrade a broad range of target proteins containing specific sequence determinants, called degrons ( 30 – 36 ). Depletion of ClpX did not impact Mrf degradation, suggesting that ClpC1 (MSMEG_6091) possibly serves as the ATPase in the process; mycobacteria do not encode a ClpA homolog.…”

“…Because ClpS plays a crucial role in target recognition by the Clp protease system ( 30 – 36 ), we hypothesized that Mrf interacts with ClpS in a zinc-dependent manner. A strain (YL16) coexpressing Mrf and ClpS 6xHis , while carrying the Atc-inducible CRISPRi system for ClpP1, was cultured in high- or low-zinc medium for 96 h, and ClpS-Mrf interaction was evaluated.…”

“…The canonical degrons for ClpS are generated upon posttranslational changes in the N-terminal sequences of target proteins ( 33 , 36 ). The N-terminal sequence of Mrf, however, remained unchanged in high- and low-zinc medium ( SI Appendix , Fig.…”