Abstract
In actinobacteria, post-translational modification of proteins with prokaryotic ubiquitin-like protein Pup targets them for degradation by a bacterial proteasome assembly consisting of the 20S core particle (CP) and the mycobacterial proteasomal ATPase (Mpa). Modification of hundreds of cellular proteins with Pup at specific surface lysines is carried out by a single Pup-ligase (PafA, proteasome accessory factor A). Pupylated substrates are recruited to the degradative pathway by binding of Pup to the N-terminal coiled-coil domains of Mpa. Alternatively, pupylation can be reversed by the enzyme Dop (deamidase of Pup). Although pupylated substrates outcompete free Pup in proteasomal degradation, potential discrimination of the degradation complex between the various pupylated substrates has not been investigated. Here we show that Mpa binds stably to an open-gate variant of the proteasome (oCP) and associates with bona fide substrates with highly similar affinities. The proteasomal degradation of substrates differing in size, structure and assembly state was recorded in real-time, showing that the pupylated substrates are processed by the Mpa-oCP complex with comparable kinetic parameters. Furthermore, the members of a complex, pupylated proteome (pupylome) purified from Mycobacterium smegmatis are degraded evenly as followed by western blotting. In contrast, analysis of the depupylation behavior of several pupylome members suggests substrate-specific differences in enzymatic turnover, leading to the conclusion that largely indiscriminate degradation competes with differentiated depupylation to control the ultimate fate of pupylated substrates.
Highlights
In mycobacteria and many other actinobacteria, covalent modification of proteins with the small (60–70 residues), intrinsically disordered protein Pup allows them to be recognized and degraded by a bacterial proteasome complex consisting of the 20S proteasome core particle and a ring-shaped ATPase referred to as mycobacterial proteasomal ATPase (Mpa) in mycobacteria or ARC (ATPase forming ring-shaped complexes) in other actinobacteria [1, 2]
Recognition of pupylated substrates by the proteasome complex occurs at the hexameric AAA+ Mpa-ring [19, 20], which is formed of three parts: one ring tier is made of the C-terminal AAA+ modules that stack on top of the 20S particle, followed N-terminally by a narrower collar-like double-ring tier formed by two consecutive β-barrel domains [21, 22]
As a recent X-ray structure of Mpa revealed the presence of a stable β-grasp domain at the very Cterminus of Mpa burying the C-terminal interaction motif of Mpa inside the Mpa central pore, it was suggested by the authors that association even with the open gate proteasome is significantly impaired by the formation of this domain [22]
Summary
In mycobacteria and many other actinobacteria, covalent modification of proteins with the small (60–70 residues), intrinsically disordered protein Pup (prokaryotic ubiquitin-like protein) allows them to be recognized and degraded by a bacterial proteasome complex consisting of the 20S proteasome core particle and a ring-shaped ATPase referred to as Mpa (mycobacterial proteasomal ATPase) in mycobacteria or ARC (ATPase forming ring-shaped complexes) in other actinobacteria [1, 2]. This proteasomal degradation pathway is not essential under standard culture conditions, it provides bacteria with a critical advantage under certain. This, contradicts earlier studies that have shown stable interaction between Mpa and oCP in vitro using size exclusion chromatography or electron microscopy [20, 26]
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