In Mycobacterium tuberculosis (Mtb) proteins post-translationally modified with prokaryotic ubiquitin-like protein (Pup, pupylation indicated as “Pup~”) can be degraded by the bacterial proteasome. While all Pup-proteasome system (PPS) enzymes are required for virulence, little is known about how their activities are regulated. In this thesis I describe how we identified a regulator, CoaX, that modulates depupylation of a specific PPS substrate, Pup~PanB, in response to pantothenate levels. As PanB, ketopantoate hydroxymethyltransferase, catalyzes the first step in pantothenate synthesis, this is the first model for regulation of PPS activity by negative feedback. CoaX and pantothenate are the first PPS regulators ever described and their discovery provides a framework for identification of additional regulators. We also took several approaches to identify pupylome degradation regulators. Multiple pieces of evidence support the hypothesis that the pupylome is degraded more when Mtb are grown on nitrate as a sole nitrogen source compared to asparagine. We purified pupylome degradation complexes under this condition and performed a transposon genetic screen to investigate of the molecular mechanisms underpinning this phenotype. While these approaches did not result in discovery of degradation regulators, they provided additional information about Mtb biology and may open up new areas for investigation. Together these findings constitute the most detailed investigation of PPS regulation to date and lay the foundation for the next era of PPS research: investigating its response, and contribution, to Mtb biology beyond protein quality control.