Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging

Abstract

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.

References

  1. 1

    Pickart, C. M. & Eddins, M. J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta 1695, 55–72 (2004)

  2. 2

    Huang, D. T., Walden, H., Duda, D. & Schulman, B. A. Ubiquitin-like protein activation. Oncogene 23, 1958–1971 (2004)

  3. 3

    Kerscher, O., Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 22, 159–180 (2006)

  4. 4

    Ciechanover, A., Elias, S., Heller, H. & Hershko, A. ‘Covalent affinity’ purification of ubiquitin-activating enzyme. J. Biol. Chem. 257, 2537–2542 (1982)

  5. 5

    Haas, A. L., Warms, J. V., Hershko, A. & Rose, I. A. Ubiquitin-activating enzyme. Mechanism and role in protein–ubiquitin conjugation. J. Biol. Chem. 257, 2543–2548 (1982)

  6. 6

    Hershko, A., Heller, H., Elias, S. & Ciechanover, A. Components of ubiquitin–protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214 (1983)

  7. 7

    Pickart, C. M. & Rose, I. A. Functional heterogeneity of ubiquitin carrier proteins. J. Biol. Chem. 260, 1573–1581 (1985)

  8. 8

    Pickart, C. M. Back to the future with ubiquitin. Cell 116, 181–190 (2004)

  9. 9

    Finley, D., Ciechanover, A. & Varshavsky, A. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell 37, 43–55 (1984)

  10. 10

    Ciechanover, A., Finley, D. & Varshavsky, A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell 37, 57–66 (1984)

  11. 11

    McGrath, J. P., Jentsch, S. & Varshavsky, A. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10, 227–236 (1991)

  12. 12

    Odorisio, T., Mahadevaiah, S. K., McCarrey, J. R. & Burgoyne, P. S. Transcriptional analysis of the candidate spermatogenesis gene Ube1y and of the closely related Ube1x shows that they are coexpressed in spermatogonia and spermatids but are repressed in pachytene spermatocytes. Dev. Biol. 180, 336–343 (1996)

  13. 13

    Lake, M. W., Wuebbens, M. M., Rajagopalan, K. V. & Schindelin, H. Mechanism of ubiquitin activation revealed by the structure of a bacterial MoeB–MoaD complex. Nature 414, 325–329 (2001)

  14. 14

    Duda, D. M., Walden, H., Sfondouris, J. & Schulman, B. A. Structural analysis of Escherichia coli ThiF. J. Mol. Biol. 349, 774–786 (2005)

  15. 15

    Lehmann, C., Begley, T. P. & Ealick, S. E. Structure of the Escherichia coli ThiS–ThiF complex, a key component of the sulfur transfer system in thiamin biosynthesis. Biochemistry 45, 11–19 (2006)

  16. 16

    Walden, H., Podgorski, M. S. & Schulman, B. A. Insights into the ubiquitin transfer cascade from the structure of the activating enzyme for NEDD8. Nature 422, 330–334 (2003)

  17. 17

    Lois, L. M. & Lima, C. D. Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1. EMBO J. 24, 439–451 (2005)

  18. 18

    Bencsath, K. P., Podgorski, M. S., Pagala, V. R., Slaughter, C. A. & Schulman, B. A. Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation. J. Biol. Chem. 277, 47938–47945 (2002)

  19. 19

    Huang, D. T. et al. Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8’s E1. Mol. Cell 17, 341–350 (2005)

  20. 20

    Huang, D. T. et al. Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity. Nature 445, 394–398 (2007)

  21. 21

    Haas, A. L. & Bright, P. M. The resolution and characterization of putative ubiquitin carrier protein isozymes from rabbit reticulocytes. J. Biol. Chem. 263, 13258–13267 (1988)

  22. 22

    Komatsu, M. et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J. 23, 1977–1986 (2004)

  23. 23

    Gu, X. et al. Cloning and characterization of a gene encoding the human putative ubiquitin conjugating enzyme E2Z (UBE2Z). Mol. Biol. Rep. (in the press)

  24. 24

    Walden, H. et al. The structure of the APPBP1–UBA3–NEDD8-ATP complex reveals the basis for selective ubiquitin-like protein activation by an E1. Mol. Cell 12, 1427–1437 (2003)

  25. 25

    Eletr, Z. M., Huang, D. T., Duda, D. M., Schulman, B. A. & Kuhlman, B. E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat. Struct. Mol. Biol. 12, 933–934 (2005)

  26. 26

    Booth, J. W., Kim, M. K., Jankowski, A., Schreiber, A. D. & Grinstein, S. Contrasting requirements for ubiquitylation during Fc receptor-mediated endocytosis and phagocytosis. EMBO J. 21, 251–258 (2002)

  27. 27

    Shringarpure, R., Grune, T., Mehlhase, J. & Davies, K. J. Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. J. Biol. Chem. 278, 311–318 (2003)

  28. 28

    Chen, X. et al. N-acetylation and ubiquitin-independent proteasomal degradation of p21Cip1. Mol. Cell 16, 839–847 (2004)

  29. 29

    Su, A. I. et al. Large-scale analysis of the human and mouse transcriptomes. Proc. Natl Acad. Sci. USA 99, 4465–4470 (2002)

  30. 30

    Su, A. I. et al. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl Acad. Sci. USA 101, 6062–6067 (2004)

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Acknowledgements

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.

Author information

Correspondence to J. Wade Harper.

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Competing interests

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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Supplementary Information 1

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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