Search

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin-like protein (Pup) that is recognized by the N-terminal coiled-coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa-proteasome complex unfolds and degrades Pup-tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N-terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.

Chaperone-proteases are responsible for the processive breakdown of proteins in eukaryotic, archaeal and bacterial cells. They are composed of a cylinder-shaped protease lined on the interior with proteolytic sites and of ATPase rings that bind to the apical sides of the protease to control substrate entry. We present a real-time FRET-based method for probing the reaction cycle of chaperone-proteases, which consists of substrate unfolding, translocation into the protease and degradation. Using this system we show that the two alternative bacterial ClpAP and ClpXP complexes share the same mechanism: after initial tag recognition, fast unfolding of substrate occurs coinciding with threading through the chaperone. Subsequent slow substrate translocation into the protease chamber leads to formation of a transient compact substrate intermediate presumably close to the chaperone-protease interface. Our data for ClpX and ClpA support the mechanical unfolding mode of action proposed for these chaperones. The general applicability of the designed FRET system is demonstrated here using in addition an archaeal PAN-proteasome complex as model for the more complex eukaryotic proteasome.