Regulated Proteolysis in Bacteria

Abstract

Regulated proteolysis is a vital process that affects all living things. Bacteria use energy-dependent AAA+ proteases to power degradation of misfolded and native regulatory proteins. Given that proteolysis is an irreversible event, specificity and selectivity in degrading substrates are key. Specificity is often augmented through the use of adaptors that modify the inherent specificity of the proteolytic machinery. Regulated protein degradation is intricately linked to quality control, cell-cycle progression, and physiological transitions. In this review, we highlight recent work that has shed light on our understanding of regulated proteolysis in bacteria. We discuss the role AAA+ proteases play during balanced growth as well as how these proteases are deployed during changes in growth. We present examples of how protease selectivity can be controlled in increasingly complex ways. Finally, we describe how coupling a core recognition determinant to one or more modifying agents is a general theme for regulated protein degradation.

Keywords: AAA+ proteases; ClpP; ClpX; Lon.

Figure 1

Energy-dependent proteases are composed of an ATP-hydrolysis active unfoldase domain and a chambered peptidase domain. Through successive rounds of ATP hydrolysis, a specific substrate protein is selected by the protease, unfolded by the ATPase domain, and translocated through a central pore to the peptidase chamber where it is degraded.

Figure 2

Proteases can survey protein quality in the cell (a) Competition between chaperones and proteases dictate the fate of proteins. Proteases, such as Lon, must be able to distinguish between normal protein dynamics with transient excursions into non-native states and terminally misfolded proteins that must be degraded before forming toxic aggregates. Lon recognizes hydrophobic motifs (shown as yellow circles) that are usually buried in the core of a native protein. These motifs are exposed more persistently for misfolded proteins than during the transient fluctuations of properly folded proteins, allowing Lon to recognize and degrade the terminally misfolded proteins. Chaperones contribute to this flux by binding misfolded proteins in an effort to refold them. (b) Following the process of trans-translation, in which a ssrA-tag is appended to incomplete polypeptides, the adaptor SspB binds tagged substrates and tethers them to ClpXP, enhancing the protease’s ability to degrade these substrates.

Figure 3

Lon is subject to allosteric regulation (a) Many proteases, such as ClpXP and ClpAP, adhere to typical Michaelis-Menten kinetics where adding increasing amounts of substrate leads to an increase in the rates of degradation until the protease becomes saturated and Vmax is reached resulting in the classic hyperbolic curve. (b) This contrasts with Lon which exhibits positive cooperativity upon increasing substrate concentration. The working model is that Lon exists as low (red triangles) and high activity (green squares) states with substrate binding promoting the highly active state. (b) In the case of substrate 1, which has a strong affinity for Lon, relatively low amounts of substrate are needed to shift Lon to the active state. (c) Substrate 2 binds Lon poorly and activation requires much higher concentrations of substrate 2. (d) If the concentration of substrate 2 is kept the same and the high affinity substrate 1 is added, Lon will be shifted to the active form, leading to robust degradation of the normally poorly degraded substrate 2.

Figure 4

Adaptors assemble in a hierarchical manner to degrade various classes of substrates. ClpXP can degrade numerous substrates on its own, included ssrA-tagged proteins. During the G1 to S transition in Caulobacter, the adaptor CpdR first primes ClpXP, allowing it to recruit the first class of substrates (PdeA, McpA, etc) for degradation. The primed protease can now recruit another adaptor, RcdA, to degrade a second class of substrates, such as the transcription factor TacA and others. Finally, the adaptor PopA binds RcdA and in the presence of the second messenger cyclic di-GMP completes the hierarchy to deliver a third class of substrates, including the master regulator CtrA, to ClpXP. As the hierarchy is assembled and adaptors are added on to the protease, specificity increases. When ClpXP is limiting, this increase in specificity also comes at the cost of preventing degradation of other members of the substrate pool.

Figure 5

Substrate degradation by ClpXP is rate-limited by the commitment step, where the protease initially engages a target, rather than the unfolding or translocation steps, which are relatively fast. Commitment is estimated to be ~ 30 seconds for degradation of tagged GFP by ClpXP (Cordova, et al. Cell 2014). Tethering adaptors (like SspB and RcdA) enhance degradation of substrate but the strength of the interaction between the adaptor and substrate must be tuned to the commitment time for the protease (regime ii). Poor adaptor-cargo binding results in failure to deliver (regime i) but binding too tightly (regime iii) hinders substrate release during the commitment step for protease engagement of the substrate.

Figure 6

Regulated proteolysis is required during physiological transitions and changes in growth (a) When bacteria are actively growing in logarithmic phase, the alternative sigma factor RpoS is rapidly degraded by ClpXP in an RssB-mediated manner. When RssB is phosphorylated, it has high affinity for RpoS and can deliver it to ClpXP for degradation. Anti-adaptors bind to RssB in different binding modes dependent on the kind of stress the bacteria is encountering, preventing it from delivering RpoS for degradation. (b) Bacillus subtilis requires proteolysis by ClpXP to ensure proper spore envelope assembly. In cells with improperly assembled envelopes, the adaptor CmpA delivers SpoIVA to ClpXP for degradation, leading to lysis of the defective cell. If the spore envelope is properly assembled, the adaptor is targeted for degradation by ClpXP instead. (c) Lon-mediated degradation is required for proper motility during the transition from liquid to solid media. In liquid media, Lon degrades SwrA with the help of SmiA, an adaptor protein. Upon shift to solid media, degradation of SwrA is inhibited, leading to an increase in SwrA levels necessary for swarming on solid media.