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
• Published: 06-07-2022
• ISSN: 2633-0679
• DOI: 10.1039/d2cb00077f

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.
Unlike most bacteria,
L. monocytogenes
encodes 2 isoforms of Caseinolytic Protease P. Balogh
et al.
show that both proteins form a heterocomplex temperature-dependently and find protein substrate candidates with an integrated proteomic approach.