Regulated Proteolysis in Bacteria: Caulobacter

Abstract

Protein degradation is essential for all living things. Bacteria use energy-dependent proteases to control protein destruction in a highly specific manner. Recognition of substrates is determined by the inherent specificity of the proteases and through adaptor proteins that alter the spectrum of substrates. In the α-proteobacterium Caulobacter crescentus, regulated protein degradation is required for stress responses, developmental transitions, and cell cycle progression. In this review, we describe recent progress in our understanding of the regulated and stress-responsive protein degradation pathways in Caulobacter. We discuss how organization of highly specific adaptors into functional hierarchies drives destruction of proteins during the bacterial cell cycle. Because all cells must balance the need for degradation of many true substrates with the toxic consequences of nonspecific protein destruction, principles found in one system likely generalize to others.

Keywords: AAA+ protease; ClpAP; ClpXP; Lon; adaptor; cell cycle.

Figure 1

A. Energy dependent AAA+ proteases must discriminate true targets from a large background of other nondegraded proteins. AAA+ proteases are composed of an ATP-dependent unfoldase and a nonspecific peptidase chamber. In vivo, specificity is principally determined by the unfoldases, which recognize substrates directly or through auxiliary proteins known as adaptors that alter specificity. B. Although these proteases differ in sequence and specificity, their core function is conserved. The unfoldase recognizes a substrate and uses cycles of ATP hydrolysis to power the unfolding of this protein. This unfolded polypeptide is concurrently translocated through a central pore to a peptidase chamber where the target is destroyed.

Figure 2

In Caulobacter, AAA+ proteases contribute to normal growth and stress responses. A. Chromosomal DNA replication requires sliding clamps that hold the polymerase to the template DNA. These clamps are loaded by an energy-dependent clamp loader, which is a complex comprised of several proteins including the ATP hydrolyzing subunit DnaX. In Caulobacter, DnaX is processed by ClpXP to form a shortened form that is required for normal growth and altering these processing dynamics reduces tolerance to DNA damage. ClpXP also degrades the SocB toxin, a sliding clamp inhibitor, which is upregulated during DNA damage. The upregulation of SocB seems to be the primary cause of cell death upon loss of ClpXP. B. Proteotoxic stress results in protein misfolding. In Caulobacter, the Lon protease rapidly degrades DnaA during stress conditions, resulting in cell cycle arrest. Reconstitution experiments support a model where buildup of misfolded proteins that are normally Lon substrates can allosterically activate the Lon protease to degrade DnaA. This protective mechanism ensures that cells wait until damage has been repaired before continuing with growth.

Figure 3

A. Levels of many proteins oscillate during the Caulobacter cell cycle. One source of control is that different classes of proteins can be degraded at different times during the cell cycle. Class I proteins are lost during the SW-ST (G1-S) transition, Class II proteins are more abundant (more stable) in ST cells, while Class III proteins are preferentially reduced in SW cells. Examples of each class are shown. See Table I for a more complete listing of proteins and proteases. There are several models for how proteins are selectively degraded during the cell cycle, including cell cycle dependent inhibition (B), changes in localization (C), or cell cycle dependent activation.

Figure 4

Levels of ClpXP substrates change during cell cycle progression, but levels of ClpXP remain constant. Purified swarmer cells are released into fresh media to initiate synchronized growth. Aliquots taken during synchronized growth are probed with antibodies against the chemoreceptor McpA, transcription factors TacA and CtrA, ClpX, and ClpP (adapted from (64)). Cell cycle-dependent phosphorylation of CpdR (58) is shown as +/-. Degradation of McpA relies only on CpdR, while TacA requires both CpdR and RcdA. CtrA additionally requires PopA for cell cycle regulated degradation.

Figure 5

Dephosphorylation of CpdR primes ClpXP for substrate recognition. A. CpdR binds directly to the N-terminal domain (NTD) of ClpX, facilitating the recognition of protease substrates (PdeA, McpA, etc). Phosphorylation of CpdR prevents binding to ClpX. Importantly, CpdR does not seem to directly bind its cargo substrates with any detectable affinity (74). B. RcdA directly binds substrates such as TacA facilitating their degradation by ClpXP protease. Here RcdA acts as a scaffolding adaptor delivering the substrate only to a protease that is first primed by CpdR. C. By contrast, canonical scaffolding adaptors, such as SspB, bind strongly to their cargo and tether substrates directly to the ClpXP protease.

Figure 6

An adaptor hierarchy regulates degradation during the cell cycle. During the developmental transition in Caulobacter that overlaps with its cell cycle, the adaptor CpdR binds the N-terminal domain (NTD) of ClpX ATPase priming (marked by pink dashed line) the protease to recruit substrates such as PdeA for degradation. The primed protease then recruits the scaffolding adaptor RcdA to degrade a range of substrates including TacA. The second messenger (cyclic di-GMP) dependent adaptor PopA binds the adaptor RcdA to deliver substrate CtrA to ClpXP protease.

Figure 7

Adaptors in other system: A. Competence development in B.subtilis. The binding of MecA adaptor to the ClpC ATPase activates the ClpCP protease. Once activated, the adaptor MecA binds and facilitates ComK degradation during exponential growth of B.subtilis. As the cells reaches to a higher density, the anti-adaptor ComS competitively inhibits ComK degradation thus stabilizing ComK for expression of competence-related genes. B. Fidelity of sporulation program in B.subtilis. In a sporulation competent cell, the adaptor CmpA is degraded, inhibiting degradation of the coat protein SpoIVA leading to the initiation of the sporulation program (117). In a sporulation defective cell, the adaptor CmpA facilitates degradation of SpoIVA, ultimately resulting in the lysis of the cell. C. ClpF-ClpS1- mediated GluTR degradation in chloroplast. ClpF and ClpS1 together form a multiprotein adaptor complex to deliver substrate GluTR to ClpCRP protease for degradation in chloroplast (91).

Figure 8

Changes in protein numbers in different cellular conditions illustrate the need for regulated protein degradation in the absence of cell division. A. Rapidly dividing cells can easily reduce protein levels by shutting of protein synthesis and diluting the protein pool through multiple cell divisions. B. By contrast, cells undergoing a developmental transition or stress response must change proteins levels in the absence of cell division. Rapid regulated protein degradation likely plays a particularly important role during these conditions.