Abstract
Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.
Highlights
Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host
In order to obtain a molecular understanding of the interactions between mycobacterial proteasome ATPase (Mpa) and the 20S proteasome as well as mechanistic insights into the engagement of pupylated substrates into the Mpa pore, we aimed to generate a substrate-engaged Mpa–20S core particle (CP) complex for study by cryo-electron microscopy (cryo-EM)
A homogeneously Pupdecorated model substrate was generated by placing the prokaryotic ubiquitinlike protein (Pup) coding sequence in-frame upstream of the gene folA encoding dihydrofolate reductase (DHFR), resulting in a linear PupDHFR fusion, in which the Pup C-terminal residue is ligated to the DHFR amino terminus through a regular peptide bond (Fig. 1a)
Summary
Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. Energy-dependent protein degradation contributes to cellular protein quality control and general protein turnover, and ensures a dynamic proteome response to changing cellular states and environments[1,2] It is carried out by compartmentalizing degradation machines consisting of a proteolytic core cylinder that must associate with ring-shaped, ATP-dependent regulator complexes in order to recruit, unfold and translocate its target protein substrates for processive degradation[3]. The bacterial 20S proteasome interacts with a ring-shaped AAA regulatory complex called the mycobacterial proteasome ATPase (Mpa) in mycobacteria or ARC (ATPase forming ring-shaped complexes) in other actinobacterial species[7,8,9,10] These bacteria evolved a unique substrate tagging and proteasome delivery pathway that is functionally analogous to ubiquitination[11,12]. While in the eukaryotic 19S regulatory particle the coiled-coil domains mediate binding of non-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
