A Dynamically Localized Protease Complex and a Polar Specificity Factor Control a Cell Cycle Master Regulator

Abstract

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.

A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression

“…Because of its multiple functions, the presence and activity of CtrA is tightly regulated, temporally and spatially, by two redundant mechanisms, synthesis-proteolysis and phosphorylation-dephosphorylation (3-5, 8-12, 22-27). Both of these mechanisms have to be simultaneously blocked to inhibit Caulobacter cell cycle progression at the G 1 ͞S transition (3,5). The selection of the right protein at the correct time in the cell cycle for its degradation by a protease depends on intrinsic recognition signals within the protein, on a tagging system, or, in some cases, on an adaptor or effector protein that participates in delivering the substrate to the protease (1).…”

“…ClpXP and its CtrA substrate are simultaneously localized to the stalked cell pole both at the swarmer-to-stalked cell transition and in the stalked cell compartment of the predivisional cell (3,8). We identify a single-domain response regulator, CpdR, that in its unphosphorylated state directs CtrA proteolysis by controlling ClpXP polar localization and activity.…”

“…Adaptor proteins that connect the targeted proteins to the proteolytic machinery provide substrate specificity (2). A recently identified degradation factor, RcdA, mediates proteolysis of the Caulobacter CtrA cell cycle master regulator by the dynamically localized ClpXP protease at specific times in the cell cycle (3). Here, we report the identification of a protein, CpdR, that directs ClpXP localization to the cell pole and show that CpdR function is under the control of a phospho-signaling pathway, thus establishing a mechanism for providing temporal and spatial specificity to bacterial proteases.…”

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication

“…every 10 min and frozen on dry ice. Labelled CtrA protein was immunoprecipitated as described by McGrath et al (2006) and resolved on a 15 % SDS-PAGE gel. HyBlot CL autoradiography films (Denville Scientific) were used.…”

“…Once a swarmer cell is exposed to sufficient nutrients, it differentiates into a stalked cell. As part of this transition, CtrA is degraded by the dynamically localizing protease ClpXP Jenal & Fuchs, 1998;McGrath et al, 2006), while DnaA continues to accumulate (Collier et al, 2006(Collier et al, , 2007. Without CtrA to block DnaA binding to the origin of replication, activated DnaA (Collier & Shapiro, 2009;Jonas et al, 2011) initiates DNA replication and activates the transcription of genes at the swarmer-to-stalked cell transition (Hottes et al, 2005).…”