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A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

Abstract

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells1. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber2. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain3,4,5,6. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base—a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro7,8,9. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6′ (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.

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Figure 1: Crosslinking strategy.
Figure 2: Specific crosslinking of Ub4 to proteasomes.
Figure 3: The 50K crosslinked protein is S6′.
Figure 4: ATP hydrolysis modulates the polyubiquitin chain–proteasome interaction (EPR).

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Acknowledgements

We thank G. DeMartino for a gift of modulator complex; A. Varshavksy for the GST–Rpt5 construct; A. Mildvan for discussions; and M. Hochstrasser and B. Cohen for comments on the manuscript. This work was supported by a grant from the National Institutes of Health. Y.A.L. is a senior fellow of the Leukemia and Lymphoma Society.

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Correspondence to Cecile M. Pickart.

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Lam, Y., Lawson, T., Velayutham, M. et al. A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal. Nature 416, 763–767 (2002). https://doi.org/10.1038/416763a

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