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The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle
Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.ORCID-id: 0000-0002-1469-4424
Antal upphovsmän: 42021 (Engelska)Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 10, artikel-id e73875Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Delta lon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

Ort, förlag, år, upplaga, sidor
2021. Vol. 10, artikel-id e73875
Nyckelord [en]
Lon protease, proteolysis, cell differentiation, flagella, FliK, bacterial cell cycle, Other
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
URN: urn:nbn:se:su:diva-198759DOI: 10.7554/eLife.73875ISI: 000711524700001PubMedID: 34693909OAI: oai:DiVA.org:su-198759DiVA, id: diva2:1612064
Tillgänglig från: 2021-11-17 Skapad: 2021-11-17 Senast uppdaterad: 2023-02-07Bibliografiskt granskad
Ingår i avhandling
1. Regulated Proteolysis: The Secrets of Lon: Cellular Roles and Stress-dependent Regulation of the Lon Protease
Öppna denna publikation i ny flik eller fönster >>Regulated Proteolysis: The Secrets of Lon: Cellular Roles and Stress-dependent Regulation of the Lon Protease
2023 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Proteolysis is crucial in cells to maintain a functional proteome. It is required for removal of damaged and unfolded proteins during protein quality control, and serves as a mechanism to regulate protein levels through regulated proteolysis. The latter targets specific proteins under certain conditions to rapidly adjust the levels of these proteins. Thereby, activity of the targeted proteins is reduced or even eliminated. Many cellular processes like cell cycle progression, differentiation and stress response/adaptation depend on proteolytic removal of regulatory proteins via proteolysis. One protease with crucial roles in both protein quality control and regulated proteolysis is called Lon and is found in many species across all domains of life.

Extensive research has revealed many aspects of proteolysis by Lon and identified several Lon substrates. The dimorphic bacterium Caulobacter crescentus is a model organisms to study proteolysis and Lon as the two cell types, a flagellated swarmer cell and a stalked sessile cell, and the transition between them heavily depends on proteolysis. However, certain details, like recognition of substrates and its regulation are largely unknown. Here I focus on regulated proteolysis by Lon in C. crescentus, specifically on novel substrates, their recognition, and the regulation of Lon.

Study I: Using quantitative proteomics of wildtype and mutant C. crescentus strains we identified potential substrates of Lon. Out of these, we focused on the stalk biogenesis regulator StaR and the flagellar hook length determination factor FliK. Both proteins are developmental regulators, whose protein levels oscillate during the cell cycle. Our experiments showed that turnover by Lon is required to maintain these oscillations and disruption thereof results in deregulation of the stalk and the flagellum.

Study II: We used proteolytically inactive Lon to co-purify interactors of Lon and identify them by mass spectrometry. Thereby, we found an uncharacterized heat shock protein that regulates the activity of Lon, and due to our findings named it LarA (Lon activity regulator A). We showed that LarA interacts with Lon at an allosteric site and modulates the activity of Lon via its C-terminal amino acids. In most cases LarA exhibits a stimulating effect on the degradation of the substrates, indicating that LarA regulates substrate specificity and guarantees efficient degradation of the affected substrates. The same residues involved in the modulating interaction also serve as a degron for degradation of LarA by Lon to shut off LarA-mediated modulation if not needed anymore.

Study III: SigT is a driver of gene expression of the general stress response in C. crescentus. It was detected in both previous studies, which indicates that it is degraded by Lon. We showed that it is a substrate of Lon in vitro. Based on steady-state levels of SigT during and after sucrose-induced stress, we could show that Lon-mediated degradation is important during the recovery. In addition, LarA-mediated regulation of Lon stimulates turnover of SigT, indicating that SigT levels are fine-tuned by LarA under certain conditions.

In summary, we identified novel regulatory roles of Lon on differentiation and stress response in C. crescentus and discovered LarA as a novel modulator of Lon activity. The results of these studies once more emphasize the importance of Lon as a regulator of various cellular processes. 

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2023. s. 63
Nyckelord
Lon protease, regulated proteolysis, allosteric regulation, Caulobacter crescentus, stress responses
Nationell ämneskategori
Biokemi och molekylärbiologi Mikrobiologi
Forskningsämne
molekylär biovetenskap
Identifikatorer
urn:nbn:se:su:diva-214634 (URN)978-91-8014-192-5 (ISBN)978-91-8014-193-2 (ISBN)
Disputation
2023-03-23, P216, NPQ-huset, Svante Arrhenius Väg 20 A, Stockholm, 13:30 (Engelska)
Opponent
Handledare
Tillgänglig från: 2023-02-28 Skapad: 2023-02-07 Senast uppdaterad: 2023-02-28Bibliografiskt granskad

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Jonas, Kristina

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Science for Life Laboratory (SciLifeLab)Institutionen för molekylär biovetenskap, Wenner-Grens institut
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