Updated project metadata. Mycobacteria and other Actinobacteria possess proteasomal degradation pathways in addition to the common bacterial compartmentalizing protease systems. Proteasomal degradation supports survival of these bacteria in adverse environments and conditions. The mycobacterial proteasome interacts with multiple ring-shaped activators, including the bacterial proteasome activator (Bpa), which facilitates the energy-independent degradation of heat shock repressor HspR in the successful human pathogen M. tuberculosis. To explore if this observation can be corroborated in the soil bacterium M. smegmatis, we performed an enrichment study in a bpa knockout in M. smegmatis. We grew the wild type M. smegmatis strain and the bpa knockout strain in biological triplicates under standard conditions or under heat shock and analyzed proteins which increased in abundance by data-independent acquisition mass spectrometry.