The chaperonin GroESL facilitatesCaulobacter crescentuscell division by supporting the function of the actin homologue FtsA

Abstract

The highly conserved chaperonin GroESL performs a crucial role in protein folding, however the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle, using the model organism Caulobacter crescentus. Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events such as DNA replication and chromosome segregation are able to continue when GroESL folding is insufficient, and find that deficiency of the bacterial actin homologue FtsA function mediates the GroESL-dependent block in cell division. Our data suggest that a GroESL-FtsA interaction is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus. In addition to supporting FtsA function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.ImportanceAll organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and FtsA, a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis, and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.

The Protein Quality Control Network in Caulobacter crescentus

“…Several mechanisms by which the highly conserved nodes of the PQC network ensure cell cycle progression during optimal conditions are known, and ongoing work has begun to reveal mechanistic insight into how these functions change when stress is encountered. Substrate trapping or depletion experiments connected to mass spectrometry have begun to characterize client proteins of the major chaperones and proteases (Bhat et al, 2013;Goemans et al, 2018a;Schroeder et al, 2020), and with this an understanding of how processes are regulated as individual proteins transit through the PQC network has become possible.…”

“…In addition to DnaKJ/E, the Caulobacter genome contains one other DnaK-like protein (CCNA_01543) and five additional DnaJ-like proteins containing the characteristic J domain (CCNA_00965, CCNA_02218, CCNA_02245, CCNA_02860, CCNA_03105); however, it is currently unknown if these proteins direct the specificity of DnaK folding toward different client protein pools (Kampinga and Craig, 2010), or if they have another role. Depletion of either DnaKJ/E or GroESL halts the Caulobacter cell cycle in distinct stages; loss of DnaKJ/E results in a block of DNA replication initiation (Jonas et al, 2013;Schramm et al, 2017), whereas depletion of GroESL results in a cell division defect (Susin et al, 2006;Schroeder et al, 2020). Mild depletion of either of these folding machines produces an increase in the other (Da Silva et al, 2003;Susin et al, 2006), suggesting some degree of compensation exists, yet neither DnaKJ/E nor GroESL can fully substitute the stress response or cell cycle functions of the other.…”

“…An overview of Caulobacter proteins whose folding state, or solubility, is influenced by GroESL has recently been described (Schroeder et al, 2020). Through this approach, cell cycleregulated proteins involved in peptidoglycan biosynthesis and cell division were identified to have an interaction with GroESL folding availability in optimal conditions, including the FtsZinteracting proteins FtsA and FzlA, which mediate a cell division block during GroESL depletion (Schroeder et al, 2020). While the role of GroESL in cell cycle progression is beginning to be understood, the contributions of this highly stress-induced folding machine during stress conditions have not yet been uncovered.…”