Evidence for an Interaction between Ubiquitin-Conjugating Enzymes and the 26S Proteasome

Abstract

The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4^C86A), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.

ACKNOWLEDGMENTS

This work was supported by grants to K.M. from the National Institutes of Health (GM52058), and the American Heart Association (9850170T). D.L. was supported by a Predoctoral Fellowship from the American Heart Association.

We thank members of the laboratory for helpful discussions and suggestions. M. Colon-Berlingeri and D. Rodriguez are thanked for their contributions during laboratory rotations. We thank J. Dohmen, M. Ellison, and M. Hochstrasser for strains, plasmids, and reagents.

ADDENDUM IN PROOF

While this paper was in review, Y. Xie and A. Varshavsky reported that E3 proteins could interact with the proteasome (Proc. Natl. Acad. Sci. USA 97:2497–2502, 2000).