The mycobacterial Mpa-proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus.
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.
Striebel
Frank
F
Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.
Hunkeler
Moritz
M
Summer
Heike
H
Weber-Ban
Eilika
E
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C479920:Mpa protein, Mycobacterium tuberculosis