Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation at elevated temperatures

• Published in: RSC chemical biology Vol. 3; no. 7; pp. 955 - 971
• Main Authors: Balogh, Dóra, Eckel, Konstantin, Fetzer, Christian, Sieber, Stephan A.
• Format: Journal Article
• Language: English
• Published: RSC 06-07-2022
• Subjects: Chemistry
• DOI: 10.1039/D2CB00077F
• EISSN: 2633-0679

Listeria monocytogenes
exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1 : 1 at 30 °C, while almost no complex formation occurred below 4 °C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed
L. monocytogenes
ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain
via
whole proteome mass-spectrometry (MS) at 37 °C and 42 °C. While the Δ
clpP1
strain only altered the expression of very few proteins, the Δ
clpP2
and Δ
clpP1/2
strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.