Updated project metadata. Tuberculosis remains a leading cause of worldwide infectious mortality and one of the top ten leading causes of death overall. While advances in public health have contributed to a reduction in tuberculosis cases, the prevalence of multidrug-resistant Mycobacterium tuberculosis (Mtb) (MDR-TB) infections has created an urgent need to exploit novel drug targets. One such target is the ClpC1P1P2 protease, which degrades folded cytosolic proteins through the cooperation of the ATP-dependent unfoldase ClpC1 and the ClpP1P2 peptidase. Both protease components are strictly essential for Mtb viability and are validated therapeutic targets. However, efforts to develop anti-Mtb compounds are constrained by a limited understanding of Clp protease function and essentiality. Thus, it is crucial to identify physiological substrates and pathways regulated by this protease. In this study, we identify cellular proteins that interact with the ClpC1 unfoldase in Mycolicibacterium smegmatis (Msm), a nonpathogenic surrogate for Mtb. Using a FLAG-tagged ClpC1 variant with mutations within the Walker B motifs, candidate ClpC1 interaction partners were captured by co-immunoprecipitation and identified by mass spectrometry-based proteomics (LC-MS/MS). Notably, our work reveals a novel proteolytic substrate, 5-oxoprolinase, which is recognized by ClpC1P1P2 via an N-terminal degradation sequence.