Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

Abstract

In this study we determine that the Not4 E3 ligase is important for proteasome integrity. Consequently, deletion of Not4 leads to an accumulation of polyubiquitinated proteins and reduced levels of free ubiquitin. In the absence of Not4, the proteasome regulatory particle (RP) and core particle (CP) form salt-resistant complexes, and all other forms of RPs are unstable. Not4 can associate with RP species present in purified proteasome holoenzyme but not with purified RP. Additionally, Not4 interacts with Ecm29, a protein that stabilizes the proteasome. Interestingly, Ecm29 is identified in RP species that are inactive and not detectable in cells lacking Not4. In the absence of Not4, Ecm29 interacts less well with the proteasome and becomes ubiquitinated and degraded. Our results characterize Ecm29 as a proteasome chaperone whose appropriate interaction with the proteasome requires Not4.

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Correction for Panasenko and Collart, “Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29”

Supplemental material for this article may be found athttp://dx.doi.org/10.1128/MCB.01210-10.

ACKNOWLEDGMENTS

We thank Daniel Finley and Jeroen Roelofs for strains and antibodies against Ecm29, Rpn5, Rpn8, and Rpn12 and for helpful discussions, Marion Schmidt for the strains, and Patrizia Arboit for the help with mass spectrometry analysis. We thank Jörg Urban for a critical reading of the manuscript.

This work was supported by grant 3100AO-100793 from the National Science Foundation to M.A.C.