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A dynamic complex of signaling proteins uses polar localization to regulate cell-fate asymmetry in Caulobacter crescentus.

ABSTRACT: Cellular asymmetry is critical to metazoan development and the life cycle of many microbes. In Caulobacter, cell cycle progression and the formation of asymmetric daughter cells depend on the polarly-localized histidine kinase CckA. How CckA is regulated and why activity depends on localization are unknown. Here, we demonstrate that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Early in the cell cycle, CckA is activated by the dephosphorylation of DivK throughout the cell. However, in later stages, when phosphorylated DivK levels are high, CckA activation relies on polar localization with a DivK phosphatase. Localization thus creates a protected zone for CckA within the cell, without the use of membrane-enclosed compartments. Our results reveal the mechanisms by which CckA is regulated in a cell-type-dependent manner. More generally, our findings reveal how cells exploit subcellular localization to orchestrate sophisticated regulatory processes.

SUBMITTER: Tsokos CG

PROVIDER: S-EPMC3068846 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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A dynamic complex of signaling proteins uses polar localization to regulate cell-fate asymmetry in Caulobacter crescentus.

Tsokos Christos G CG Perchuk Barrett S BS Laub Michael T MT

Developmental cell 20110301 3

Cellular asymmetry is critical to metazoan development and the life cycle of many microbes. In Caulobacter, cell cycle progression and the formation of asymmetric daughter cells depend on the polarly-localized histidine kinase CckA. How CckA is regulated and why activity depends on localization are unknown. Here, we demonstrate that the unorthodox kinase DivL promotes CckA activity and that the phosphorylated regulator DivK inhibits CckA by binding to DivL. Early in the cell cycle, CckA is activ ...[more]

PMID: 21397844

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Project description:In Caulobacter crescentus, timely degradation of several proteins by the ClpXP protease is critical for proper cell cycle progression. During the cell cycle, the ClpXP protease, the substrate CtrA, and many other proteins are localized to the stalked pole dependent on a polar interaction hub composed of PopZ protein oligomers. Prior work suggests that the localization of ClpXP, protease substrates, and cofactors is needed for recognition of substrates, such as CtrA, by ClpXP. Here, we formally test this hypothesis by examining the role of PopZ in ClpXP activity and find, surprisingly, that CtrA degradation is enhanced in cells lacking polar localization due to loss of PopZ. The ClpXP adaptor CpdR is required for this enhanced degradation of CtrA and other adaptor-dependent substrates, but adaptor-independent substrate degradation is not affected upon loss of PopZ. We find that overexpression of PopZ also leads to faster degradation of CtrA but is likely due to nonphysiologically relevant recognition of CtrA by ClpXP alone, as loss of CpdR does not affect this enhancement. Our main conclusion is that loss of PopZ, and therefore loss of polar localization, does not result in the loss of ClpXP-regulated proteolysis, as would be predicted from a model which requires polar localization of ClpXP for its activation. Rather, our data point to a model where PopZ normally restrains ClpXP proteolysis by promoting the inactivation of the CpdR adaptor, perhaps through the activity and localization of the CckA kinase.IMPORTANCE Regulated proteolysis is critical for the cell cycle progression of bacteria, such as Caulobacter crescentus According to one model, this regulated proteolysis requires localization of the ClpXP protease at the stalked pole for its subsequent degradation of substrates, such as CtrA. This study offers evidence that supports an alternative model to explain how localization might influence protein degradation. Using a delocalized in vivo system created by the deletion of a polar organizing protein, PopZ, we show that activation of the ClpXP protease is independent of its polar localization. The data point to a role for PopZ in restraining ClpXP activity, likely by controlling the activity of upstream regulators of protease activity, such as CckA, though changes in its localization.

Project description:Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria.IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

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A dynamic complex of signaling proteins uses polar localization to regulate cell-fate asymmetry in Caulobacter crescentus.

Publications

A dynamic complex of signaling proteins uses polar localization to regulate cell-fate asymmetry in Caulobacter crescentus.

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