Identification of substrates of the Mycobacterium tuberculosis proteasome and characterization of a ubiquitin-like protein that signals their degradation

The putative proteasome-associated proteins Mpa ( Mycobacterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. In this work we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase (FabD) and ketopantoate hydroxymethyltransferase (PanB), enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological amounts of these enzymes required Mpa, PafA and proteasome protease activity. It was unknown how these substrates were being targeted for proteasome-mediated degradation, as prokaryotes lack ubiquitin (Ub), the degradation signal for the eukaryotic proteasome. Further analysis led to the identification of a prokaryotic ubiquitin-like protein, Pup (Rv2111c), which was specifically conjugated to these substrates in Mtb. Pupylation occurred on lysines and required PafA. Pupylated proteins were degraded in an Mpa-dependent manner and a lack of pupylation led to an increased stability of proteasome substrates, thereby connecting pupylation with degradation. Although analogous to ubiquitylation, pupylation appears to proceed by a different chemistry. Thus, like eukaryotes, bacteria may use a small protein modifier to control protein stability.