Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals Stephen C. Smitha, Kamal K. Joshib,1, Justin J. Zika,1, Katherine Trinha, Aron Kamajayaa, Peter Chienb,2, and Kathleen R. Ryana,2 aDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720; and bProgram in Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003 Edited by Sue Wickner, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved August 8, 2014 (received for review April 29, 2014) The cell-division cycle of Caulobacter crescentus depends on peri- Although ClpX alone recognizes some substrates, the degrada- odic activation and deactivation of the essential response regula- tion of other substrates requires or is enhanced by adaptor proteins tor CtrA. Although CtrA is critical for transcription during some (9). For example, the Escherichia coli protein SspB is an adaptor parts of the cell cycle, its activity must be eliminated before chro- for the degradation of substrates with a C-terminal ssrA peptide mosome replication because CtrA also blocks the initiation of DNA (10), which arise during failed translation events (11). SspB binds replication. CtrA activity is down-regulated both by dephosphory- to ClpX and to the ssrA peptide, increasing the local effective lation and by proteolysis, mediated by the ubiquitous ATP-dependent substrate concentration through tethering (12, 13). Adaptors protease ClpXP. Here we demonstrate that proteins needed for themselves can be tightly regulated; for example, the response rapid CtrA proteolysis in vivo form a phosphorylation-dependent regulator RssB acts as an adaptor for the rapid ClpXP-mediated and cyclic diguanylate (cdG)-dependent adaptor complex that proteolysis of the stationary phase sigma factor RpoS during ex- accelerates CtrA degradation in vitro by ClpXP. The adaptor com- ponential growth in E. coli (14). In stationary phase or in response plex includes CpdR, a single-domain response regulator; PopA, to specific stresses, up-regulation of antiadaptor proteins that block MICROBIOLOGY a cdG-binding protein; and RcdA, a protein whose activity cannot RssB from binding RpoS drives a surge in RpoS levels (15, 16). be predicted. When CpdR is unphosphorylated and when PopA is In the Caulobacter cell cycle, CtrA proteolysis is spatially and bound to cdG, they work together with RcdA in an all-or-none temporally restricted such that it occurs only during the G1–S K manner to reduce the m of CtrA proteolysis 10-fold. We further transition, either during the development of a motile swarmer identified a set of amino acids in the receiver domain of CtrA that cell into a sessile stalked cell, or in the stalked compartment of modulate its adaptor-mediated degradation in vitro and in vivo. the late predivisional cell (Fig. 1; ref. 17). In addition to ClpX Complex formation between PopA and CtrA depends on these and ClpP, which are present throughout the cell cycle (7), the amino acids, which reside on alpha-helix 1 of the CtrA receiver accessory proteins RcdA, CpdR, and PopA, and the small mol- domain, and on cdG binding by PopA. These results reveal that ecule cyclic di-GMP (cdG), are required for the rapid degrada- each accessory factor plays an essential biochemical role in the regu- tion of CtrA in vivo (18–20). lated proteolysis of CtrA and demonstrate, to our knowledge, the RcdA is conserved in some alpha-proteobacteria, and its tran- first example of a multiprotein, cdG-dependent proteolytic adaptor. scription is regulated directly by CtrA in Caulobacter (18). RcdA exists as a dimer, with each monomer comprising a three-helix he alpha-proteobacterium Caulobacter crescentus is a pow- Terful model organism for understanding cell polarity, dif- Significance ferentiation, and cell-cycle regulation in bacteria (1). Each Caulobacter cell division is asymmetric, yielding a motile swarmer cell (SW) and a sessile stalked cell (ST). The stalked Controlled degradation of specific proteins is used by all cell immediately initiates chromosome replication and enters organisms to change cell behavior in response to internal or a new division cycle, whereas the swarmer cell must first differ- external cues. Because ATP-dependent proteases, such as ClpXP, entiate into a stalked cell before undergoing cell division (Fig. 1). have a broad range of targets, accessory proteins called adaptors An elaborate network of two-component signaling proteins are often necessary for selective substrate proteolysis. Here we orchestrates the Caulobacter cycle of division and development show that three proteins work together as a multicomponent adaptor to stimulate the degradation of a key regulatory pro- (2). The essential DNA-binding response regulator CtrA is at the Caulobacter crescentus center of this regulatory scheme, activating or repressing the tein, CtrA, in . The adaptor is only func- tional when one of the components, CpdR, is unphosphorylated transcription of >100 genes that are required for cell division, and when another component, PopA, is bound to the signaling motility, DNA methylation, and other processes (3). Because molecule cyclic diguanylate. These features ensure that CtrA is CtrA also inhibits the initiation of chromosome replication (4, only proteolyzed during a specific window in the Caulobacter 5), its activity must be temporarily eliminated at the G1–S cell- cell-division cycle. cycle transition for DNA replication and the subsequent cell division to occur. Inactivation of CtrA is achieved through two Author contributions: S.C.S., K.K.J., J.J.Z., K.T., A.K., P.C., and K.R.R. designed research; redundant mechanisms: dephosphorylation (6) and proteolysis by S.C.S., K.K.J., J.J.Z., K.T., A.K., and K.R.R. performed research; S.C.S., K.K.J., J.J.Z., A.K., P.C., theproteaseClpXP(7). and K.R.R. analyzed data; and P.C. and K.R.R. wrote the paper. ClpXP is a highly conserved ATP-dependent protease crucial The authors declare no conflict of interest. for protein quality control and in regulated degradation of key This article is a PNAS Direct Submission. substrates to implement developmental switches and adapt to 1K.K.J. and J.J.Z. contributed equally to this work. external cues (8). The barrel-shaped oligomer of ClpP contains 2To whom correspondence may be addressed. Email: [email protected] or pchien@ hydrolysis active sites on its inner surface. To access these sites, biochem.umass.edu. substrates are recognized, unfolded, and translocated into the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ClpP chamber by a ring-shaped hexamer of the ATPase ClpX. 1073/pnas.1407862111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1407862111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 second messengers to promote robust degradation of an es- sential cell-cycle regulator, reconciling in vitro and cellular SW ST PD ClpXP observations of CtrA degradation. CpdR Results RcdA Identification of Degradation-Enhancing Amino Acids in the CtrA Receiver Domain. CtrA contains two internal signals that de- CtrA termine its pattern of cell cycle-dependent proteolysis. One de- PopA-cdG terminant consists of two alanine residues at the C terminus, which are required for direct recognition by ClpX (17, 26, 28), Caulobacter and the other signal resides somewhere in the first 56 amino Fig. 1. CtrA dynamics during the cell cycle. SW, swarmer cell; acids of the CtrA receiver domain (25). We reasoned that amino ST, stalked cell; PD, predivisional cell. Straight lines represent pili. Wavy lines represent flagella. Filled circles denote nonreplicating chromosomes. Theta acids in CtrA that are important for degradation would be structures denote replicating chromosomes. Red and pink shading represent coconserved with rcdA and cpdR, which are found in ∼1/3 of CtrA protein. sequenced α-proteobacteria (29). We therefore aligned CtrA protein sequences from genomes in 26 different genera of the α-proteobacteria (Fig. S1; ref. 30) and identified residues in the bundle connected by disordered loops and a disordered C-ter- first half of the CtrA receiver domain that are highly conserved minal peptide necessary for regulated CtrA proteolysis (21). in genomes containing homologs of rcdA and cpdR, but that are CpdR is a single-domain response regulator that is phosphory- divergent in bacteria that lack these genes. We then mapped lated by the histidine kinase CckA and the histidine phospho- these residues onto a model of the CtrA receiver domain based transferase ChpT, which also act on CtrA (22). However, CpdR on the structure of the response regulator Spo0F (Protein Data must be unphosphorylated to promote CtrA degradation (19). Bank ID code 1NAT; ref. 31). Six of the residues we identified This signaling logic dictates that when CtrA is phosphorylated are predicted to lie on the exposed surface of the first alpha-helix and active, it will also be stabilized due to the phosphorylation of (α1) of CtrA (S10, A11, Q14, K21, S22, and E23), whereas two of CpdR. Before S phase, when both are dephosphorylated, the the residues are predicted to be partially or completely buried in activity and stability of CtrA are concomitantly reduced (6, 19). the interior of the protein (F25 and G40; Fig. 2A). PopA is composed of two tandem receiver domains and a Based on this analysis, we examined the in vivo degradation C-terminal diguanylate cyclase (DGC) domain. The DGC domain pattern of a chimeric protein containing segments of CtrA and of PopA is inactive, but cdG binding at an allosteric site is required CzcR, a nondegradable CtrA ortholog (25). CtrA–RD+15, for PopA to stimulate CtrA proteolysis in vivo (20). PopA is which contains the receiver domain and the last 15 amino acids thought to link CtrA proteolysis to cellular cdG levels, which of CtrA, but lacks the DNA-binding domain, has the same cell- transiently increase during swarmer cell differentiation (23, 24).