Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging


Modification of proteins with ubiquitin or ubiquitin-like proteins (UBLs) by means of an E1–E2–E3 cascade controls many signalling networks1,2,3. Ubiquitin conjugation involves adenylation and thioesterification of the carboxy-terminal carboxylate of ubiquitin by the E1-activating enzyme Ube1 (Uba1 in yeast), followed by ubiquitin transfer to an E2-conjugating enzyme through a transthiolation reaction4,5,6,7. Charged E2s function with E3s to ubiquitinate substrates1. It is currently thought that Ube1/Uba1 is the sole E1 for charging of E2s with ubiquitin in animals and fungi1,8. Here we identify a divergent E1 in vertebrates and sea urchin, Uba6, which specifically activates ubiquitin but not other UBLs in vitro and in vivo. Human Uba6 and Ube1 have distinct preferences for E2 charging in vitro, and their specificity depends in part on their C-terminal ubiquitin-fold domains, which recruit E2s. In tissue culture cells, Uba6 is required for charging a previously uncharacterized Uba6-specific E2 (Use1), whereas Ube1 is required for charging the cell-cycle E2s Cdc34A and Cdc34B. Our data reveal unexpected complexity in the pathways that control the conjugation of ubiquitin, in which dual E1s orchestrate the charging of distinct cohorts of E2s.

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Figure 1: Uba6 activates ubiquitin in vitro.
Figure 2: Uba6 activates ubiquitin in vivo.
Figure 3: Systematic analysis of E2-conjugating enzymes for targets of Uba6.
Figure 4: Distinct requirements for charging of the ubiquitin conjugating enzymes Use1 and Cdc34 in vivo.


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We thank D. Finley, B. Tansey, S. Elledge, J. Lou, R. Mulligan and B. Schulman for technical assistance, reagents and/or discussions, and B. Schulman and A. Sali for assistance with Modeller software. This work was supported by grants from the National Institutes of Health to J.W.H. and to S.P.G.

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Correspondence to J. Wade Harper.

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Sequences for human Uba6 and Use1 have been deposited in the GenBank database under accession numbers EF623992 and EF623993, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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This file contains Supplementary Notes, Supplementary Methods, Supplementary Figures S1-S7 with Legends, Supplementary Table S1 and additional references. (PDF 7147 kb)

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