Summary Regulated protein degradation is essential. The timed destruction of crucial proteins by the ClpXP protease drives cell-cycle progression in the bacterium Caulobacter crescentus. Although ClpXP is active alone, additional factors are inexplicably required for cell-cycle dependent proteolysis. Here, we show that these factors constitute an adaptor hierarchy where different substrates are destroyed based on the degree of adaptor assembly. The hierarchy builds upon priming of ClpXP by the adaptor CpdR, which promotes degradation of one class of substrates and also recruits the adaptor RcdA to degrade a second class of substrates. Adding the PopA adaptor promotes destruction of a third class of substrates, while inhibiting degradation of the second class. We dissect RcdA to generate bespoke adaptors, identifying critical substrate elements needed for RcdA recognition and uncovering additional cell-cycle dependent ClpXP substrates. Our work reveals how hierarchical adaptors and primed proteases orchestrate regulated proteolysis during bacterial cell-cycle progression.

Significance Controlled degradation of specific proteins is used by all organisms to change cell behavior in response to internal or external cues. Because ATP-dependent proteases, such as ClpXP, have a broad range of targets, accessory proteins called adaptors are often necessary for selective substrate proteolysis. Here we show that three proteins work together as a multicomponent adaptor to stimulate the degradation of a key regulatory protein, CtrA, in Caulobacter crescentus . The adaptor is only functional when one of the components, CpdR, is unphosphorylated and when another component, PopA, is bound to the signaling molecule cyclic diguanylate. These features ensure that CtrA is only proteolyzed during a specific window in the Caulobacter cell-division cycle.

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1-S transition involves the degradation of an essential DNA-binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1-S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1-S transition so that cells can properly activate CtrA-dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade a critical cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programs of gene expression critical to polar morphogenesis and cell division.

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 will likely generalize to others.

Multiple strategies have been developed to facilitate the efficient production of bispecific IgG (BsIgG) in single host cells. For example, we previously demonstrated near quantitative (≥90%) formation of BsIgG of different species and isotypes by combining 'knob-into-hole' mutations for heavy chain heterodimerization with engineered antigen-binding fragments (Fabs) for preferential cognate heavy/light chain pairing. Surprisingly, in this study we found high yield (>65%) of BsIgG 1 without Fab engineering to be a common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both C H 1/C L interfaces was sufficient for near quantitative yield (>90%) of BsIgG 1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, we demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG 1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG 1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG 1. This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.