Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E ( pafE ) Mutant

Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled byMycobacterium tuberculosis p roteasomea ccessoryf actorE (PafE). Recently, we found that aMycobacterium tuberculosis pafE mutant has slowed growthin vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (h eats hockp roteinr epressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterizedpafE suppressor mutants that grew similarly topafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of apafE mutant were caused primarily by the inability of the proteasome to degrade HspR.IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence ofM. tuberculosis . In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.