Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates

The protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup). Proteasome accessory factor A (PafA) attaches Pup to proteins to target them for degradation by the proteasome. Free Pup is unstable and never observed in extracts of M. tuberculosis, an observation that led us to hypothesize that PafA may need alternative sources of Pup. Here, we show that PafA can move Pup from one proteasome substrate, inositol 1-phosphate synthetase (Ino1), to two different proteins, malonyl coenzyme A (CoA)-acyl carrier protein transacylase (FabD) and lonely guy (Log). This apparent “transpupylation” reaction required a previously unrecognized depupylase activity in PafA, and, surprisingly, this depupylase activity was much more efficient than the activity of the dedicated depupylase Dop (deamidase of Pup). Thus, PafA can potentially use both newly synthesized Pup and recycled Pup to doom proteins for degradation.