ABSTRACTBacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with non-polar and/or structurally unconstrained C-termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we investigated if the C-termini of two non-homologous CtpA substrates are important for their degradation. We determined that these substrates have extended C-termini, compared to their closest E. coli homologs. Removing seven amino acids from these extensions was sufficient to inhibit their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C-terminus from the other, but not by adding an unrelated sequence. However, modification of the C-terminus of non-substrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C-termini of CtpA substrates are required but not sufficient for degradation. Although C-terminal truncated substrates were not degraded, they still associated with the LbcA•CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C-terminal-independent interaction with the LbcA•CtpA complex, followed by C-terminal-dependent degradation, perhaps because CtpA must initiate cleavage at a specific C-terminal site.IMPORTANCECarboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that degradation by CtpA requires at least two separable phenomena, and that one of them depends on information encoded in the substrate C-terminus. A C-terminal-independent association with the LbcA•CtpA complex is followed by C-terminal-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.