Characterization of the Proteasome Accessory Factor (paf) Operon inMycobacterium tuberculosis

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Abstract
In a previous screen forMycobacterium tuberculosismutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with theM. tuberculosisproteasome were identified. One of these genes,pafA(proteasomeaccessoryfactor A), encodes a protein of unknown function. In this work, we determined thatpafAis in an operon with two additional genes,pafBandpafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions inpafBandpafC. In contrast to thepafAmutant, thepafBandpafCmutants were not severely sensitized to RNI, butpafBandpafCwere nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein inM. tuberculosis. Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.