Ubiquitin-like protein activation by e1 enzymes: the apex for downstream signalling pathways

KEY POINTS * At the start of each ubiquitin-like protein (UBL) cascade is an E1 enzyme, which activates the UBL and then directs the UBL to downstream pathways. UBL proteins and E1 (or

activating) enzymes have their origins in prokaryotic biosynthetic pathways. * In humans, eight E1 enzymes are known to initiate UBL conjugation. We refer to E1s for ubiquitin (UBA1 and UBA6

(also known as UBE1L2)), SUMO (SAE1–UBA2), NEDD8 (NAE1–UBA3) and ISG15 (UBA7) as canonical, owing to their related domain structures and enzymatic mechanisms, and to the more divergent E1s

for URM1 (UBA4), UFM1 (UBA5) and ATG12 and ATG8 isoforms (ATG7) as non-canonical. (ATG is autophagy-related protein; ISG15 is interferon-stimulated gene 15; NAE1 is NEDD8-activating enzyme

1; SAE1 is SUMO-activating enzyme 1; UBA is ubiquitin-activating enzyme; UFM1 is ubiquitin-fold modifier 1; URM1 is ubiquitin-related modifier 1). * Canonical E1 structures all have an

adenylation domain that resembles prokaryotic ancestors, and two domains that are specific to canonical E1s: a catalytic Cys domain that contains the Cys that is involved in the E1∼UBL

thioester linkage and a carboxyl-terminal ubiquitin-fold domain that resembles ubiquitin and that binds E2. * In addition to their chemical roles in initiating UBL conjugation cascades, E1s

also establish specificity, by matching a particular UBL with only cognate E2s. Rules for how this specificity is achieved are only beginning to emerge, but it is clear that specificity is

achieved at multiple levels. * A particularly intriguing non-canonical E1 is UBA4 (MOCS3 in humans). Recent data indicate that UBA4 initiates a sulphur transfer pathway, in a manner related

to bacterial E1-like enzymes. * E1s are unique in the UBL conjugating pathway in that they are the only components that use ATP, and this property is being exploited to develop selective

inhibitors. The potential for the use of such inhibitors in therapy is high, given the links seen between components of UBL cascades and human disease. ABSTRACT Attachment of ubiquitin or

ubiquitin-like proteins (known as UBLs) to their targets through multienzyme cascades is a central mechanism to modulate protein functions. This process is initiated by a family of

mechanistically and structurally related E1 (or activating) enzymes. These activate UBLs through carboxy-terminal adenylation and thiol transfer, and coordinate the use of UBLs in specific

downstream pathways by charging cognate E2 (or conjugating) enzymes, which then interact with the downstream ubiquitylation machinery to coordinate the modification of the target. A broad

understanding of how E1 enzymes activate UBLs and how they selectively coordinate UBLs with downstream function has come from enzymatic, structural and genetic studies. Access through your

institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print

issues and online access $209.00 per year only $17.42 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to

local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT

BEING VIEWED BY OTHERS RING DOMAINS ACT AS BOTH SUBSTRATE AND ENZYME IN A CATALYTIC ARRANGEMENT TO DRIVE SELF-ANCHORED UBIQUITINATION Article Open access 22 February 2021 STRUCTURES OF UBA6

EXPLAIN ITS DUAL SPECIFICITY FOR UBIQUITIN AND FAT10 Article Open access 15 August 2022 BREAKING THE K48-CHAIN: LINKING UBIQUITIN BEYOND PROTEIN DEGRADATION Article 16 February 2024

ACCESSION CODES ACCESSIONS PROTEIN DATA BANK * 1JWA * 1R4M * 1U9B * 1Y8R * 1Y8X * 2AYZ * 2NVU * 3CMM CHANGE HISTORY * _ 09 APRIL 2009 In the main text, 'inorganic phosphate' in the

following two sentences should read 'inorganic pyrophosphate': "The basic side chains of MoaD or ThiS would stabilize the developing negative charge from the ensuing

pentacovalent phosphate intermediate, allowing the generation of MoaD∼adenylate or ThiS∼adenylate and inorganic phosphate." And: "Progression of the cascade is driven by the

release of the small molecule products inorganic phosphate and AMP in the first and second steps, respectively56,57,58,62,63." _ REFERENCES * Ciechanover, A. Intracellular protein

degradation: from a vague idea, through the lysosome and the ubiquitin–proteasome system, and onto human diseases and drug targeting (Nobel lecture). _Angew. Chem. Int. Edn Engl._ 44,

5944–5967 (2005). Article  CAS  Google Scholar  * Goldberg, A. L. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. _Biochem. Soc. Trans._ 35,

12–17 (2007). Article  CAS  PubMed  Google Scholar  * 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). DESCRIBES THE CHROMATOGRAPHIC SEPARATION OF E1, E2 AND E3 ACTIVITIES AND DEMONSTRATES RECONSTITUTION OF

THE FIRST UBIQUITIN LIGASE REACTION. Article  CAS  PubMed  Google Scholar  * Hochstrasser, M. Lingering mysteries of ubiquitin-chain assembly. _Cell_ 124, 27–34 (2006). Article  PubMed 

Google Scholar  * Pickart, C. M. & Fushman, D. Polyubiquitin chains: polymeric protein signals. _Curr. Opin. Chem. Biol._ 8, 610–616 (2004). Article  CAS  PubMed  Google Scholar  *

Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. _Nature Rev. Mol. Cell Biol._ 6, 610–621 (2005). Article  CAS  Google Scholar  * Hochstrasser, M. Ubiquitin

signalling: what's in a chain? _Nature Cell Biol._ 6, 571–572 (2004). Article  CAS  PubMed  Google Scholar  * Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. _Biochem.

J._ 399, 361–372 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Harper, J. W. & Schulman, B. A. Structural complexity in ubiquitin recognition. _Cell_ 124, 1133–1136

(2006). Article  CAS  PubMed  Google Scholar  * Ciechanover, A. Proteolysis: from the lysosome to ubiquitin and the proteasome. _Nature Rev. Mol. Cell Biol._ 6, 79–87 (2005). Article  CAS 

Google Scholar  * Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell-division cycle (Nobel lecture). _Angew. Chem. Int. Edn Engl._ 44,

5932–5943 (2005). Article  CAS  Google Scholar  * Hochstrasser, M. Biochemistry. All in the ubiquitin family. _Science_ 289, 563–564 (2000). Article  CAS  PubMed  Google Scholar  *

Hochstrasser, M. Evolution and function of ubiquitin-like protein-conjugation systems. _Nature Cell Biol._ 2, E153–E157 (2000). Article  CAS  PubMed  Google Scholar  * Kerscher, O.,

Felberbaum, R. & Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. _Annu. Rev. Cell Dev. Biol._ 22, 159–180 (2006). Article  CAS  PubMed  Google Scholar

  * Pickart, C. M. & Eddins, M. J. Ubiquitin: structures, functions, mechanisms. _Biochim. Biophys. Acta_ 1695, 55–72 (2004). Article  CAS  PubMed  Google Scholar  * Varshavsky, A.

Regulated protein degradation. _Trends Biochem. Sci._ 30, 283–286 (2005). Article  CAS  PubMed  Google Scholar  * Hershko, A., Ciechanover, A. & Varshavsky, A. The ubiquitin system.

_Nature Med._ 6, 1073–1081 (2000). Article  CAS  PubMed  Google Scholar  * Yeh, E. T., Gong, L. & Kamitani, T. Ubiquitin-like proteins: new wines in new bottles. _Gene_ 248, 1–14 (2000).

Article  CAS  PubMed  Google Scholar  * Pan, Z. Q., Kentsis, A., Dias, D. C., Yamoah, K. & Wu, K. Nedd8 on cullin: building an expressway to protein destruction. _Oncogene_ 23,

1985–1997 (2004). Article  CAS  PubMed  Google Scholar  * Yamoah, K. et al. Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail. _Proc. Natl

Acad. Sci. USA_ 105, 12230–12235 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Duda, D. M. et al. Structural insights into NEDD8 activation of cullin–RING ligases:

conformational control of conjugation. _Cell_ 134, 995–1006 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Saha, A. & Deshaies, R. J. Multimodal activation of the

ubiquitin ligase SCF by Nedd8 conjugation. _Mol. Cell_ 32, 21–31 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Geiss-Friedlander, R. & Melchior, F. Concepts in

sumoylation: a decade on. _Nature Rev. Mol. Cell Biol._ 8, 947–956 (2007). Article  CAS  Google Scholar  * Wang, C., Xi, J., Begley, T. P. & Nicholson, L. K. Solution structure of ThiS

and implications for the evolutionary roots of ubiquitin. _Nature Struct. Biol._ 8, 47–51 (2001). Article  PubMed  CAS  Google Scholar  * 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). DESCRIBES THE STRUCTURE OF THE MOEB–MOAD

COMPLEX AND REVEALS THE MECHANISM OF MOAD ADENYLATION. THIS IS THE ONLY STRUCTURE TO DATE OF AN E1-LIKE ENZYME WITH AN ADENYLATE. Google Scholar  * Taylor, S. V. et al. Thiamin biosynthesis

in _Escherichia coli_. Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation. _J. Biol. Chem._ 273, 16555–16560 (1998). Article  CAS  PubMed  Google

Scholar  * Leimkuhler, S., Wuebbens, M. M. & Rajagopalan, K. V. Characterization of _Escherichia coli_ MoeB and its involvement in the activation of molybdopterin synthase for the

biosynthesis of the molybdenum cofactor. _J. Biol. Chem._ 276, 34695–34701 (2001). Article  CAS  PubMed  Google Scholar  * Duda, D. M., Walden, H., Sfondouris, J. & Schulman, B. A.

Structural analysis of _Escherichia coli_ ThiF. _J. Mol. Biol._ 349, 774–786 (2005). Article  CAS  PubMed  Google Scholar  * 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). Article  CAS  PubMed  Google Scholar  *

Sloper-Mould, K. E., Jemc, J. C., Pickart, C. M. & Hicke, L. Distinct functional surface regions on ubiquitin. _J. Biol. Chem._ 276, 30483–30489 (2001). Article  CAS  PubMed  Google

Scholar  * Roush, R. F., Nolan, E. M., Lohr, F. & Walsh, C. T. Maturation of an _Escherichia coli_ ribosomal peptide antibiotic by ATP-consuming N–P bond formation in microcin C7. _J_.

_Am_. _Chem. Soc._ 130, 3603–3609 (2008). Article  CAS  Google Scholar  * Pearce, M. J., Mintseris, J., Ferreyra, J., Gygi, S. P. & Darwin, K. H. Ubiquitin-like protein involved in the

proteasome pathway of _Mycobacterium tuberculosis_. _Science_ 322, 1104–1107 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * McGrath, J. P., Jentsch, S. & Varshavsky, A.

UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. _EMBO J._ 10, 227–236 (1991). Article  CAS  PubMed  PubMed Central  Google Scholar  * Handley, P. M., Mueckler, M.,

Siegel, N. R., Ciechanover, A. & Schwartz, A. L. Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1. _Proc. Natl Acad. Sci. USA_ 88, 258–262

(1991). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hatfield, P. M., Callis, J. & Vierstra, R. D. Cloning of ubiquitin activating enzyme from wheat and expression of a

functional protein in _Escherichia coli_. _J. Biol. Chem._ 265, 15813–15817 (1990). Article  CAS  PubMed  Google Scholar  * Liakopoulos, D., Doenges, G., Matuschewski, K. & Jentsch, S. A

novel protein modification pathway related to the ubiquitin system. _EMBO J._ 17, 2208–2214 (1998). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gong, L. & Yeh, E. T.

Identification of the activating and conjugating enzymes of the NEDD8 conjugation pathway. _J. Biol. Chem._ 274, 12036–12042 (1999). Article  CAS  PubMed  Google Scholar  * Desterro, J. M.,

Rodriguez, M. S., Kemp, G. D. & Hay, R. T. Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. _J. Biol. Chem._ 274, 10618–10624 (1999).

Article  CAS  PubMed  Google Scholar  * Gong, L., Li, B., Millas, S. & Yeh, E. T. Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating

enzyme complex. _FEBS Lett._ 448, 185–189 (1999). Article  CAS  PubMed  Google Scholar  * Johnson, E. S., Schwienhorst, I., Dohmen, R. J. & Blobel, G. The ubiquitin-like protein Smt3p is

activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. _EMBO J._ 16, 5509–5519 (1997). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yuan, W. & Krug, R. M.

Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. _EMBO J._ 20, 362–371 (2001). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Furukawa, K., Mizushima, N., Noda, T. & Ohsumi, Y. A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes. _J. Biol. Chem._ 275,

7462–7465 (2000). Article  CAS  PubMed  Google Scholar  * Komatsu, M. et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. _EMBO J._ 23, 1977–1986 (2004). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Jin, J., Li, X., Gygi, S. P. & Harper, J. W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. _Nature_

447, 1135–1138 (2007). TOGETHER WITH REFERENCES 45 AND 46, THIS PAPER IDENTIFIES UBA6 AS A SECOND ACTIVATING ENZYME FOR UBIQUITIN, AND IDENTIFIES USE1 AS A SPECIFIC E2 CONJUGATING ENZYME FOR

UBA6. Article  CAS  PubMed  Google Scholar  * Pelzer, C. et al. UBE1L2, a novel E1 enzyme specific for ubiquitin. _J. Biol. Chem._ 282, 23010–23014 (2007). Article  CAS  PubMed  Google

Scholar  * Chiu, Y. H., Sun, Q. & Chen, Z. J. E1-L2 activates both ubiquitin and FAT10. _Mol. Cell_ 27, 1014–1023 (2007). IDENTIFIES UBA6 AS A CANDIDATE E1 FOR FAT10. Article  CAS 

PubMed  Google Scholar  * Mizushima, N. et al. A protein conjugation system essential for autophagy. _Nature_ 395, 395–398 (1998). Article  CAS  PubMed  Google Scholar  * Huang, D. T.,

Walden, H., Duda, D. & Schulman, B. A. Ubiquitin-like protein activation. _Oncogene_ 23, 1958–1971 (2004). Article  CAS  PubMed  Google Scholar  * 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). DESCRIBES THE FIRST CRYSTAL STRUCTURE OF THE

CANONICAL E1 PROTEIN COMPLEX RESPONSIBLE FOR ACTIVATING NEDD8, AND DEFINES THE DOMAIN STRUCTURE OF CANONICAL E1S. Article  CAS  PubMed  Google Scholar  * 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). DESCRIBES THE FIRST STRUCTURE OF THE HETERODIMERIC SUMO

ACTIVATING ENZYME BOUND TO SUMO. Article  CAS  PubMed  PubMed Central  Google Scholar  * Lee, I. & Schindelin, H. Structural insights into E1-catalyzed ubiquitin activation and transfer

to conjugating enzymes. _Cell_ 134, 268–278 (2008). DESCRIBES THE FIRST STRUCTURE OF AN ACTIVATING ENZYME FOR UBIQUITIN IN COMPLEX WITH UBIQUITIN. Article  CAS  PubMed  Google Scholar  *

Komatsu, M. et al. The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1–E2 complex formation. _J. Biol. Chem._ 276, 9846–9854

(2001). Article  CAS  PubMed  Google Scholar  * Tanida, I., Tanida-Miyake, E., Ueno, T. & Kominami, E. The human homolog of _Saccharomyces cerevisiae_ Apg7p is a protein-activating

enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. _J. Biol. Chem._ 276, 1701–1706 (2001). Article  CAS  PubMed  Google Scholar  * Schmitz, J. et al. The

sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins. _Biochemistry_ 47, 6479–6489 (2008). Article  CAS 

PubMed  Google Scholar  * Ichimura, Y. et al. A ubiquitin-like system mediates protein lipidation. _Nature_ 408, 488–492 (2000). Article  CAS  PubMed  Google Scholar  * Haas, A. L. &

Rose, I. A. The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. _J. Biol. Chem._ 257, 10329–10337 (1982). Article  CAS  PubMed  Google Scholar  * 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). DEMONSTRATES THAT UBA1

BECOMES DOUBLY LOADED WITH UBIQUITIN, WITH ONE MOLECULE IN A THIOESTER WITH THE CATALYTIC CYS OF UBA1 AND THE SECOND UBIQUITIN AS AN ADENYLATE. Article  CAS  PubMed  Google Scholar  * Haas,

A. L., Warms, J. V. & Rose, I. A. Ubiquitin adenylate: structure and role in ubiquitin activation. _Biochemistry_ 22, 4388–4394 (1983). Article  CAS  PubMed  Google Scholar  *

Ciechanover, A., Heller, H., Katz-Etzion, R. & Hershko, A. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system. _Proc. Natl Acad. Sci. USA_ 78, 761–765

(1981). SUGGESTS A TWO STEP MECHANISM FOR UBIQUITIN ACTIVATION, THE FIRST INVOLVING ADENYLATION AND THE SECOND INVOLVING THIOESTER FORMATION. Article  CAS  PubMed  PubMed Central  Google

Scholar  * Ciechanover, A., Elias, S., Heller, H. & Hershko, A. “Covalent affinity” purification of ubiquitin-activating enzyme. _J. Biol. Chem._ 257, 2537–2542 (1982). DESCRIBES THE

PURIFICATION OF E1 UBIQUITIN ACTIVATING ENZYME, UBA1, WHICH USED THE THIOESTER AS A TOOL FOR RECOVERY OF ACTIVE ENZYME. THE ABILITY TO PURIFY ACTIVE UBA1 REVOLUTIONIZED THE FIELD. Article 

CAS  PubMed  Google Scholar  * Pickart, C. M. & Rose, I. A. Functional heterogeneity of ubiquitin carrier proteins. _J. Biol. Chem._ 260, 1573–1581 (1985). DEMONSTRATED THE EXISTENCE OF

A FAMILY OF E2 CONJUGATING ENZYMES THAT ARE CHARGED BY UBA1, FORESHADOWING THE DISCOVERY THAT DISTINCT E2S ARE INVOLVED IN DISTINCT BIOLOGICAL PATHWAYS. Article  CAS  PubMed  Google Scholar

  * 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).

Article  CAS  PubMed  Google Scholar  * Haas, A. L., Bright, P. M. & Jackson, V. E. Functional diversity among putative E2 isozymes in the mechanism of ubiquitin–histone ligation. _J.

Biol. Chem._ 263, 13268–13275 (1988). Article  CAS  PubMed  Google Scholar  * Pickart, C. M., Kasperek, E. M., Beal, R. & Kim, A. Substrate properties of site-specific mutant ubiquitin

protein (G76A) reveal unexpected mechanistic features of ubiquitin-activating enzyme (E1). _J. Biol. Chem._ 269, 7115–7123 (1994). Article  CAS  PubMed  Google Scholar  * Tokgoz, Z.,

Bohnsack, R. N. & Haas, A. L. Pleiotropic effects of ATP..Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme. _J. Biol. Chem._ 281, 14729–14737 (2006). Article  PubMed 

CAS  Google Scholar  * Bohnsack, R. N. & Haas, A. L. Conservation in the mechanism of Nedd8 activation by the human AppBp1–Uba3 heterodimer. _J. Biol. Chem._ 278, 26823–26830 (2003).

Article  CAS  PubMed  Google Scholar  * 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). Article  CAS  PubMed  Google Scholar  * Huang, D. T. et al. Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity. _Nature_ 445, 394–398

(2007). REPORTS THE FIRST STRUCTURE OF A DOUBLY LOADED CANONICAL E1 ENZYME, AND DEFINES A THIOESTER SWITCH RESPONSIBLE FOR PROMOTING TRANSFER OF THE UBIQUITIN LIKE PROTEIN NEDD8 TO ITS E2,

UBC12. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhao, C. et al. The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-α/β-induced ubiquitin-like protein.

_Proc. Natl Acad. Sci. USA_ 101, 7578–7582 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Huang, D. T. et al. A unique E1–E2 interaction required for optimal conjugation of

the ubiquitin-like protein NEDD8. _Nature Struct. Mol. Biol._ 11, 927–935 (2004). Article  CAS  Google Scholar  * 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). Article  CAS  PubMed  Google Scholar  * Souphron, J. et al. Structural dissection of a gating mechanism preventing

misactivation of ubiquitin by NEDD8's E1. _Biochemistry_ 47, 8961–8969 (2008). Article  CAS  PubMed  Google Scholar  * Wilkinson, K. D. et al. A specific inhibitor of the ubiquitin

activating enzyme: synthesis and characterization of adenosyl-phospho-ubiquitinol, a nonhydrolyzable ubiquitin adenylate analogue. _Biochemistry_ 29, 7373–7380 (1990). Article  CAS  PubMed 

Google Scholar  * Madden, M. M. et al. Substrate properties of ubiquitin carboxyl-terminally derived peptide probes for protein ubiquitination. _Biochemistry_ 47, 3636–3644 (2008). Article 

CAS  PubMed  Google Scholar  * Szczepanowski, R. H., Filipek, R. & Bochtler, M. Crystal structure of a fragment of mouse ubiquitin-activating enzyme. _J. Biol. Chem._ 280, 22006–22011

(2005). Article  CAS  PubMed  Google Scholar  * Sullivan, M. L. & Vierstra, R. D. Cloning of a 16-kDa ubiquitin carrier protein from wheat and _Arabidopsis thaliana_. Identification of

functional domains by _in vitro_ mutagenesis. _J. Biol. Chem._ 266, 23878–23885 (1991). Article  CAS  PubMed  Google Scholar  * 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). Article  CAS  PubMed  Google

Scholar  * Durfee, L. A., Kelley, M. L. & Huibregtse, J. M. The basis for selective E1–E2 interactions in the ISG15 conjugation system. _J. Biol. Chem._ 283, 23895–23902 (2008). Article

  CAS  PubMed  PubMed Central  Google Scholar  * Lee, T. V. et al. The E1 ubiquitin-activating enzyme Uba1 in _Drosophila_ controls apoptosis autonomously and tissue growth non-autonomously.

_Development_ 135, 43–52 (2008). Article  CAS  PubMed  Google Scholar  * 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. _Nature Struct. Mol. Biol._ 12, 933–934 (2005). Article  CAS  Google Scholar  * Reverter, D. & Lima, C.

D. Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex. _Nature_ 435, 687–692 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rabut, G. &

Peter, M. Function and regulation of protein neddylation. 'Protein modifications: beyond the usual suspects' review series. _EMBO Rep._ 9, 969–976 (2008). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Tatham, M. H. et al. Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation. _Biochemistry_ 42, 9959–9969 (2003). Article  CAS 

PubMed  Google Scholar  * Whitby, F. G., Xia, G., Pickart, C. M. & Hill, C. P. Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway

enzymes. _J. Biol. Chem._ 273, 34983–34991 (1998). Article  CAS  PubMed  Google Scholar  * Wang, J. et al. The intrinsic affinity between E2 and the Cys domain of E1 in ubiquitin-like

modifications. _Mol. Cell_ 27, 228–237 (2007). Article  PubMed  PubMed Central  CAS  Google Scholar  * Haas, A. L. Structural insights into early events in the conjugation of ubiquitin and

ubiquitin-like proteins. _Mol. Cell_ 27, 174–175 (2007). Article  CAS  PubMed  Google Scholar  * Huang, D. T., Zhuang, M., Ayrault, O. & Schulman, B. A. Identification of conjugation

specificity determinants unmasks vestigial preference for ubiquitin within the NEDD8 E2. _Nature Struct. Mol. Biol._ 15, 280–287 (2008). Article  CAS  Google Scholar  * Finley, D.,

Ciechanover, A. & Varshavsky, A. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. _Cell_ 37, 43–55 (1984). IDENTIFIES UBA1 AS THE PROTEIN MUTATED

IN THE TS85 CELL LINE AND DEMONSTRATES THAT UBA1 IS RESPONSIBLE FOR THE VAST MAJORITY OF UBIQUITIN CONJUGATION IN MAMMALIAN CELLS. Article  CAS  PubMed  Google Scholar  * Ciechanover, A.,

Finley, D. & Varshavsky, A. Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin–protein conjugation. _Prog. Clin. Biol. Res._ 180, 17–31 (1985). CAS

  PubMed  Google Scholar  * Schlabach, M. R. et al. Cancer proliferation gene discovery through functional genomics. _Science_ 319, 620–624 (2008). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Strous, G. J., van Kerkhof, P., Govers, R., Ciechanover, A. & Schwartz, A. L. The ubiquitin conjugation system is required for ligand-induced endocytosis and

degradation of the growth hormone receptor. _EMBO J._ 15, 3806–3812 (1996). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rocca, A., Lamaze, C., Subtil, A. & Dautry-Varsat, A.

Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor β chain to late endocytic compartments. _Mol. Biol. Cell_ 12, 1293–1301 (2001). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Swanson, R. & Hochstrasser, M. A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in _Saccharomyces cerevisiae_. _FEBS

Lett._ 477, 193–198 (2000). Article  CAS  PubMed  Google Scholar  * Ghaboosi, N. & Deshaies, R. J. A conditional yeast E1 mutant blocks the ubiquitin–proteasome pathway and reveals a

role for ubiquitin conjugates in targeting Rad23 to the proteasome. _Mol. Biol. Cell_ 18, 1953–1963 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kulkarni, M. & Smith,

H. E. E1 ubiquitin-activating enzyme UBA-1 plays multiple roles throughout _C. elegans_ development. _PLoS Genet._ 4, e1000131 (2008). Article  PubMed  PubMed Central  CAS  Google Scholar  *

Pfleger, C. M., Harvey, K. F., Yan, H. & Hariharan, I. K. Mutation of the gene encoding the ubiquitin activating enzyme Uba1 causes tissue overgrowth in _Drosophila_. _Fly (Austin)_ 1,

95–105 (2007). Article  Google Scholar  * Nollen, E. A. et al. Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. _Proc. Natl

Acad. Sci. USA_ 101, 6403–6408 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ramser, J. et al. Rare missense and synonymous variants in UBE1 are associated with X-linked

infantile spinal muscular atrophy. _Am. J. Hum. Genet._ 82, 188–193 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A. &

Schwartz, A. L. The ubiquitin-activating enzyme E1 is phosphorylated and localized to the nucleus in a cell cycle-dependent manner. _J. Biol. Chem._ 271, 15608–15614 (1996). Article  CAS 

PubMed  Google Scholar  * Nouspikel, T. & Hanawalt, P. C. Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme. _Proc. Natl

Acad. Sci. USA_ 103, 16188–16193 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Grenfell, S. J., Trausch-Azar, J. S., Handley-Gearhart, P. M., Ciechanover, A. &

Schwartz, A. L. Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent. _Biochem. J._ 300, 701–708 (1994). Article  CAS  PubMed  PubMed Central  Google Scholar

  * Dohmen, R. J. et al. An essential yeast gene encoding a homolog of ubiquitin-activating enzyme. _J. Biol. Chem._ 270, 18099–18109 (1995). Article  CAS  PubMed  Google Scholar  * Bossis,

G. & Melchior, F. Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. _Mol. Cell_ 21, 349–357 (2006). Article  CAS  PubMed  Google Scholar  * Boggio, R.,

Colombo, R., Hay, R. T., Draetta, G. F. & Chiocca, S. A mechanism for inhibiting the SUMO pathway. _Mol. Cell_ 16, 549–561 (2004). Article  CAS  PubMed  Google Scholar  * Boggio, R.,

Passafaro, A. & Chiocca, S. Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation. _J. Biol. Chem._ 282, 15376–15382 (2007). Article  CAS  PubMed  Google

Scholar  * Handeli, S. & Weintraub, H. The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1. _Cell_ 71, 599–611 (1992).

Article  CAS  PubMed  Google Scholar  * Chen, Y., McPhie, D. L., Hirschberg, J. & Neve, R. L. The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S–M

checkpoint and causes apoptosis in neurons. _J. Biol. Chem._ 275, 8929–8935 (2000). Article  CAS  PubMed  Google Scholar  * Kumar, A. et al. Commensal bacteria modulate cullin-dependent

signaling via generation of reactive oxygen species. _EMBO J._ 26, 4457–4466 (2007). Article  CAS  PubMed  PubMed Central  Google Scholar  * Goehring, A. S., Rivers, D. M. & Sprague, G.

F. Jr. Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. _Eukaryot. Cell_ 2, 930–936 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Schlieker, C., Van der Veen, A. G., Damon, J. R., Spooner, E. & Ploegh, H. L. A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway.

_Proc. Natl Acad. Sci. USA_ 105, 18255–18260 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Leidel, S. et al. Ubiquitin-related modifier Urm1 acts as a sulphur carrier in

thiolation of eukaryotic transfer RNA. _Nature_ 458, 228–232 (2009). Article  CAS  PubMed  Google Scholar  * Nakai, Y., Nakai, M. & Hayashi, H. Thio-modification of yeast cytosolic tRNA

requires a ubiquitin-related system that resembles bacterial sulfur transfer systems. _J. Biol. Chem._ 283, 27469–27476 (2008). Article  CAS  PubMed  Google Scholar  * Huang, B., Lu, J.

& Bystrom, A. S. A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in _Saccharomyces cerevisiae_. _RNA_ 14,

2183–2194 (2008). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tanida, I. et al. Apg7p/Cvt2p: a novel protein-activating enzyme essential for autophagy. _Mol. Biol. Cell_ 10,

1367–1379 (1999). Article  CAS  PubMed  PubMed Central  Google Scholar  * Yamada, Y. et al. The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that

mediates Atg8 lipidation. _J. Biol. Chem._ 282, 8036–8043 (2007). Article  CAS  PubMed  Google Scholar  * Mizushima, T. et al. Crystal structure of Ufc1, the Ufm1-conjugating enzyme.

_Biochem. Biophys. Res. Commun._ 362, 1079–1084 (2007). Article  CAS  PubMed  Google Scholar  * Sekizawa, R. et al. Panepophenanthrin, from a mushroom strain, a novel inhibitor of the

ubiquitin-activating enzyme. _J. Nat. Prod._ 65, 1491–1493 (2002). Article  CAS  PubMed  Google Scholar  * Yang, Y. et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of

potential cancer therapeutics. _Cancer Res._ 67, 9472–9481 (2007). Article  CAS  PubMed  Google Scholar  * Tong, H., Hateboer, G., Perrakis, A., Bernards, R. & Sixma, T. K. Crystal

structure of murine/human Ubc9 provides insight into the variability of the ubiquitin-conjugating system. _J. Biol. Chem._ 272, 21381–21387 (1997). Article  CAS  PubMed  Google Scholar  *

Worthylake, D. K., Prakash, S., Prakash, L. & Hill, C. P. Crystal structure of the _Saccharomyces cerevisiae_ ubiquitin-conjugating enzyme Rad6 at 2.6 Å resolution. _J. Biol. Chem._ 273,

6271–6276 (1998). Article  CAS  PubMed  Google Scholar  * Soucy, T. A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. _Nature_ (in the press). * Huang,

D.T. et al. E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. _Mol. Cell._ 33, 483–495 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  Download

references ACKNOWLEDGEMENTS We thank the National Institutes of Health (J.W.H. and B.A.S.) and Millennium Pharmaceuticals, Inc. (J.W.H.) for funding our work on E1 enzymes and C. Regni for

assistance with figure 2. B.A.S. is an Investigator of the Howard Hughes Medical Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Departments of Structural Biology, Howard Hughes

Medical Institute, and Genetics and Tumour Cell Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, USA. brenda.schulman@stjude.org, Brenda

A. Schulman * Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. wade_harper@hms.harvard.edu, J. Wade Harper Authors * Brenda A.

Schulman View author publications You can also search for this author inPubMed Google Scholar * J. Wade Harper View author publications You can also search for this author inPubMed Google

Scholar ETHICS DECLARATIONS COMPETING INTERESTS Brenda A. Schulman is a consultant for Millennium Pharmaceuticals. J. Wade Harper is also a consultant for and receives grant support from

Millennium Pharmaceuticals. RELATED LINKS RELATED LINKS DATABASES PROTEIN DATA BANK 1JWA 1R4M 1U9B 1Y8R 1Y8X 2AYZ 2NVU 3CMM FURTHER INFORMATION Brenda A. Schulman's homepage J. Wade

Harper's homepage GLOSSARY * Adenylation The synthesis of a phosphodiester bond between a hydroxyl group and the phosphate group of AMP. In the case of ubiquitin-like protein (UBL)

conjugation cascades, the hydroxyl is from the carboxyl terminus of the UBL. * Thioester bond Covalent linkage of a sulphur with an acyl group. In the case of ubiquitin-like protein (UBL)

cascades, the Cys sulphur of an enzyme is linked to the terminal carbon of a UBL. * 26S proteasome A large multisubunit protease complex that selectively degrades polyubiquitylated proteins.

It contains a 20S particle that carries the catalytic activity and two regulatory 19S particles. * UBL fold A structural motif, also known as βGRASP fold, that has a domain with a structure

of two β-sheets, followed by an α-helix and two additional β-sheets. * Rhodanese homology domain A domain that shares sequence and structural homology to rhodanese, a mitochondrial enzyme

that catalyses Cys-mediated sulphur transfer reactions. * PP loop ATPase domain A structural motif found in a family of conserved ATP-binding proteins that serve as tRNA-modifying enzymes

and activate the tRNA through adenylation. * Elongator complex An enzyme complex that replaces the hydrogen atom on position 5 of uridine at nucleotide 34 of tRNAs by a methoxycarbonylmethyl

group. * Thiouridinylation An enzymatic process in which the 2' oxygen of uridine in the anticodon of a tRNA is replaced by sulphur. RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Schulman, B., Wade Harper, J. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. _Nat Rev Mol Cell Biol_ 10,

319–331 (2009). https://doi.org/10.1038/nrm2673 Download citation * Published: 08 April 2009 * Issue Date: May 2009 * DOI: https://doi.org/10.1038/nrm2673 SHARE THIS ARTICLE Anyone you share

the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative