A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

Donatis, Gian Marco M. De; Singh, Satyendra Kumar; Viswanathan, Sarada; Maurizi, Michael R.

doi:10.1074/jbc.m109.053736

Cited by 20 publications

(29 citation statements)

References 47 publications

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“…Multiple ClpSsubstrate complexes can dock on the N-domains of a single ClpA hexamer (23)(24)(25). As established previously, an NTE of at least 14 amino acids is necessary for ClpS to deliver an N-degron substrate ( Fig.…”

Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

“…1A) (23); however, whether the NTE acts in cis to deliver the substrate bound to its own ClpS molecule or in trans to activate delivery of a substrate bound to another ClpS molecule is unknown. The optimal ratio of ClpS to ClpA hexamer in the delivery complex has not been established, but many ratios yield functional complexes (6,23,24).To test whether the ClpS NTE acts in cis or in trans, we monitored delivery of substrates by mixtures of ClpS variants with a full-length functional NTE or with a truncated nonfunctional NTE (ClpS Δ13 ) and a WT or M40A (ClpS M40A ) N-degron-binding pocket. The ClpS M40A variant recognizes β-branched (Val and Ile) residues, termed *N-degrons, in addition to the natural E. coli N-degrons Tyr, Leu, Phe, and Trp (Fig.…”

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confidence: 99%

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Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Rivera-Rivera¹,

Roman-Hernandez²,

Sauer³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrateClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex. We establish that ClpS promotes delivery only in cis, as demonstrated by mixing ClpS variants with distinct substrate specificity and either active or inactive NTE truncations. Importantly, we find that ClpA engagement of the ClpS NTE is crucial for ClpS-mediated substrate delivery by using ClpS variants carrying "blocking" elements that prevent the NTE from entering the pore. These results support models in which enzymatic activity of ClpA actively remodels ClpS to promote substrate transfer, and highlight how ATPase/motor activities of AAA+ proteases can be critical for substrate selection as well as protein degradation.AAA+ ATPase adaptor | N-degron substrate selection | adaptor remodeling | AAA+ unfoldase/translocase A AA+ molecular machines power cellular processes as diverse as protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication, with the common theme that macromolecules are actively remodeled (1-3). Furthermore, protein quality control in all organisms involves deployment of ATPdependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel (3, 4). Adaptor proteins are known to aid recognition and degradation of certain substrates (5-8), but how enzyme-adaptor pairs ensure proper substrate selection is poorly understood.In prokaryotes and eukaryotes, the N-end rule pathway governs degradation of proteins with specific N-terminal amino acids (9, 10). In Escherichia coli, the primary destabilizing N-degron amino acids are Phe, Tyr, Trp, and Leu (11,12). ClpS, a widespread bacterial adaptor, recognizes and delivers N-degron substrates to the ClpAP or ClpCP AAA+ proteases (6,11,13). These enzymes consist of the AAA+ ClpA or ClpC unfoldases coaxially stacked with the ClpP peptidase (14-16). In eukaryotes, a family of E3 ligases shares homology with the substrate-binding region of ClpS (17, 18). These ligases recognize N-degron substrates and promote ubiquitination, which then targets the modified protein to the 26S proteasome (17,18).Multiple crystal structures reveal the regions of ClpS that bind to the N-degron, as well as a patch that binds the N-terminal domain of . This bivalent binding to the substrate and the enzyme tethers N-degron substrates to ClpAP. Tethering alone is insufficient for ClpS to promote substrate delivery however, given that deletion of 12 amino acids of the ClpS unstructured N-terminal extension (NTE; residues 1-25 in E. coli ClpS) (Fig. 1A) prevents N-degron substrate degradation, but does not block format...

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Rivera-Rivera¹,

Roman-Hernandez²,

Sauer³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Protein Purification and Quantification-GFP-SsrA (8) and LR-GFP VENUS (26) were expressed and purified as described. Wild-type ClpS protein was purified from MG1655 ⌬ara ompT cells carrying a pBAD33-clpS plasmid (18).…”

Section: Methodsmentioning

confidence: 99%

“…ClpS, an 11-kDa monomer, forms a one-to-one complex with the N-domain of the ClpA subunit (18,19). When a ClpS-substrate complex encounters the ClpA hexamer, the binding of the complex is enhanced by interaction of the flexible N-terminal peptide of ClpS with a site near or within the axial channel of the hexamer (26,27). Because steric constraints allow only one ClpS N-terminal peptide to access the axial site, this interaction not only increases binding affinity of the complex but also ensures that only one substrate molecule is positioned for delivery through the axial channel of ClpA to ClpP.…”

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confidence: 99%

The N-degradome of Escherichia coli

Humbard¹,

Surkov²,

Donatis³

et al. 2013

Journal of Biological Chemistry

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Background: Understanding of the prokaryotic N-end rule is incomplete with respect to generation of primary and secondary N-degrons. Results: Proteomics analysis of ClpS-interacting proteins identified Ͼ100 new putative N-end rule substrates in Escherichia coli. Conclusion: Both primary and secondary N-degrons are generated by limited endoproteolytic cleavage of native proteins. Significance: A possible mechanism for the generation of N-end rule substrates is proposed.

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The N‐end rule pathway and regulation by proteolysis

2011

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The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing N a -terminally acetylated (Nt-acetylated) residues. The Arg/Nend rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed Ac N-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Ntamidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.

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A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

Cited by 20 publications

References 47 publications

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

The N-degradome of Escherichia coli

The N‐end rule pathway and regulation by proteolysis

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