Identification of a depupylation regulator for an essential enzyme in Mycobacterium tuberculosis

Mycobacterium tuberculosis can use a proteasome to degrade proteins when they are post-translationally modified with prokaryotic ubiquitin-like protein (Pup). While pupylation is reversible, mechanisms regulating depupylation have not been identified. Here, we identify a depupylation regulator, CoaX, a pseudo-pantothenate kinase. Pantothenate synthesis enzymes were more abundant in a ?coaX mutant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome system-dependent manner. In vitro, CoaX accelerated depupylation of Pup~PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup~PanB in response to pantothenate levels. Overall design: RNA was extracted from quadruplicate biological cultures of H37Rv pMV strep ev (WT with an empty vector, MHD761), coaX::hyg pMV strep ev (?coaX with an empty vector, MHD1845), and coaX::hyg pMV strep coaX (complemented strain, MHD1846) of Mycobacterium tuberculosis (10 OD equivalents).