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J. Biol. Chem., Vol. 282, Issue 52, 37454-37460, December 28, 2007

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Direct Catalysis of Lysine 48-linked Polyubiquitin Chains by the Ubiquitin-activating Enzyme^*

J. Torin Huzil¹, Rajeet Pannu², Christopher Ptak³, Grace Garen, and Michael J. Ellison⁴

From the Department of Biochemistry and Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Within the ubiquitin degradation pathway, the canonical signal is a lysine 48-linked polyubiquitin chain that is assembled upon an internal lysine residue of a substrate protein. Once constructed, this ubiquitin chain becomes the principle signal for recognition and target degradation by the 26S proteasome. The mechanism by which polyubiquitin chains are assembled on a substrate protein, however, has yet to be clearly defined. In an in vitro model system, purified E2-ubiquitin thiolester was unable to catalyze the formation of polyubiquitin chains in the absence of the ubiquitin-activating enzyme E1. Mutagenesis of key residues within the E1 active site revealed that its conserved catalytic cysteine residue is essential for the formation of these chains. Moreover, inactivation of the E2 active site had no effect on the ability of E1 to catalyze ubiquitin chain formation. These findings strongly suggest E1 is responsible for not only the activation of ubiquitin but also for the direct catalytic extension of a lysine 48-linked polyubiquitin chain.

Received for publication, June 26, 2007 , and in revised form, October 17, 2007.

^* This research was supported by the National Cancer Institute of Canada and the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ Present address: Dept. of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada.

² Present address: Regan Campbell Ward-McCann, New York, NY, 10017.

³ Present address: Dept. of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada.

⁴ To whom correspondence should be addressed: 3-67 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. E-mail: michael.ellison{at}ualberta.ca.

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