Adaptor-Dependent Degradation of a Cell-Cycle Regulator Uses a Unique Substrate Architecture

Rood, Keith L.; Clark, Nathaniel E.; Stoddard, Patrick R.; Garman, S.C.; Chien, Peter

doi:10.1016/j.str.2012.04.019

Cited by 28 publications

(35 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…SUMO-tagged DnaX variants, ClpP, and ClpX were purified following procedures as before (37). Standard degradation reactions were performed in ClpXP degradation buffer [20 mM Hepes, 100 mM KCl, 10 mM MgCl2, 10% (vol/vol) glycerol, pH 7.5, and a creatine kinasebased ATP regeneration mix] and analyzed as before (37).…”

Section: Experimental Methodsmentioning

confidence: 99%

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Vass

Chien

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions. protease stalling | ClpP | ClpX internal recognition

show abstract

Section: Experimental Methodsmentioning

confidence: 99%

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Vass

Chien

2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…These secondary recognition signals can also be used to direct the unfoldase to a specific oligomeric or conformational state of the substrate, and thus serve a regulatory function. Adaptor-mediated degradation can also be influenced by “anti-adaptor” proteins or post-translational modification to enhance or inhibit protease recognition 51–53 .…”

Section: Bacterial Aaa+ Proteasesmentioning

confidence: 99%

Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines

2015

View full text Add to dashboard Cite

Preface To maintain protein homeostasis, AAA+ proteolytic machines degrade damaged and unneeded proteins in bacteria, archaea and eukaryotes. This process involves ATP-dependent unfolding of a target protein and subsequent translocation into a self-compartmentalized proteolytic chamber. Related AAA+ enzymes also disaggregate and remodel proteins. Recent structural and biochemical studies, in combination with direct visualization of unfolding and translocation in single-molecule experiments, have illuminated molecular mechanisms and suggest how remodelling of macromolecular complexes by AAA+ enzymes could occur without global denaturation. In this Review, we discuss the structural and mechanistic features of AAA+ proteases and remodeling machines, focusing on bacterial ClpXP and ClpX as paradigms. We also consider the potential of these enzymes as antibacterial targets and outline future challenges for the field.

show abstract

“…These observations provide a strong link between CbrA-dependent regulation of CtrA stability and free-living cell cycle outcomes. However, C. crescentus CpdR promotes the regulated degradation of multiple ClpXP targets, including the 54 -dependent response regulator TacA (53), the c-di-GMP phosphodiesterase PdeA (39,54), and CtrA (33). It is therefore possible that CpdR1-dependent degradation of a target other than CtrA is responsible for CpdR1 D53A suppression of ⌬cbrA mutant phenotypes.…”

Section: Discussionmentioning

confidence: 99%

Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1

Schallies¹,

Sadowski²,

Meng³

et al. 2015

J. Bacteriol.

Self Cite

View full text Add to dashboard Cite

CbrA is a DivJ/PleC-like histidine kinase of DivK that is required for cell cycle progression and symbiosis in the alphaproteobacterium Sinorhizobium meliloti. Loss of cbrA results in increased levels of CtrA as well as its phosphorylation. While many of the known Caulobacter crescentus regulators of CtrA phosphorylation and proteolysis are phylogenetically conserved within S. meliloti, the latter lacks the PopA regulator that is required for CtrA degradation in C. crescentus. In order to investigate whether CtrA proteolysis occurs in S. meliloti, CtrA stability was assessed. During exponential growth, CtrA is unstable and therefore likely to be degraded in a cell cycle-regulated manner. Loss of cbrA significantly increases CtrA stability, but this phenotype is restored to that of the wild type by constitutive ectopic expression of a CpdR1 variant that cannot be phosphorylated (CpdR1 D53A ). Addition of CpdR1 D53A fully suppresses cbrA mutant cell cycle defects, consistent with regulation of CtrA stability playing a key role in mediating proper cell cycle progression in S. meliloti. Importantly, the cbrA mutant symbiosis defect is also suppressed in the presence of CpdR1 D53A . Thus, regulation of CtrA stability by CbrA and CpdR1 is associated with free-living cell cycle outcomes and symbiosis. IMPORTANCEThe cell cycle is a fundamental process required for bacterial growth, reproduction, and developmental differentiation. Our objective is to understand how a two-component signal transduction network directs cell cycle events during free-living growth and host colonization. The Sinorhizobium meliloti nitrogen-fixing symbiosis with plants is associated with novel cell cycle events. This study identifies a link between the regulated stability of an essential response regulator, free-living cell cycle progression, and symbiosis. Sinorhizobium meliloti is a member of the class Alphaproteobacteria that grows free-living in the soil or as a beneficial nitrogen-fixing symbiont in association with legumes in the genera Medicago, Melilotus, and Trigonella. As a free-living organism, S. meliloti undergoes an asymmetric cell division with once-andonly-once DNA replication per cell cycle (1, 2, 3). However, inside its host, this bacterium undergoes differentiation into a bacteroid that includes a novel cell cycle program of repeated DNA replication in the absence of cell division (endoreduplication) (3). The underlying molecular mechanisms that dictate cell cycle progression and differentiation in S. meliloti remain to be explored in detail and therefore represent a novel aspect of symbiont physiology. Known and putative S. meliloti cell cycle regulators are conserved among other members of the Alphaproteobacteria that also specialize in chronic colonization of eukaryotic hosts, in particular rhizobial species in the genera Agrobacterium, Bartonella, and Brucella (2, 4). Thus, a deeper understanding of S. meliloti cell cycle regulation will provide insight into processes that are broadly important to both cell cycle ...

show abstract

Adaptor-Dependent Degradation of a Cell-Cycle Regulator Uses a Unique Substrate Architecture

Cited by 28 publications

References 47 publications

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus

Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines

Sinorhizobium meliloti CtrA Stability Is Regulated in a CbrA-Dependent Manner That Is Influenced by CpdR1

Contact Info

Product

Resources

About