Mechanism of Lysine 48-Linked Ubiquitin-Chain Synthesis by the Cullin-RING Ubiquitin-Ligase Complex SCF-Cdc34

Petroski, Matthew D.; Deshaies, Raymond J.

doi:10.1016/j.cell.2005.09.033

Cited by 254 publications

(362 citation statements)

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“…It had been presumed that polyubiquitin chains are formed by conjugating ubiquitin moieties, one at a time, first to a lysine residue in a substrate, and then to a lysine in the previously-attached ubiquitin molecule. This appears to be the case for some E2s (13)(14)(15). However, we and several other groups recently found that ubiquitin chains can also be preassembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its yeast homologue Ubc7p both in vitro and in vivo (16)(17)(18)(19).…”

mentioning

confidence: 65%

“…This was caused by the transfer of Flag-UbK48R to free ubiquitin present in the reaction. The formation of such a diubiquitin species has been used as an indicator of ligase activity for some RING E3s, and the amount of diubiquitin generated depends on the concentration of the ubiquitin acceptor (13,27). Indeed, when the Ube2g2ϳFlag-UbK48R thioester was incubated with excess ubiquitin, gp78C⌬L could generate diubiquitin more efficiently than wild-type gp78C (Fig.…”

Section: Results Gp78 Contains 2 Oligomerization Sites One Of Which mentioning

confidence: 99%

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Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

115

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Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78 -Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.crystallography ͉ endoplasmic reticulum-associated protein degradation ͉ gp78 ͉ polyubiquitin chain C ovalent attachment of the 76-residue ubiquitin to polypeptides regulates the stability, localization, or activity of the modified proteins (1-3). This modification requires concerted actions of 3 types of enzymes: an activating enzyme (E1) that forms a thioester linkage (designated herein as ''ϳ'') between its catalytic cysteine and the carboxyl group of the Gly-76 in ubiquitin; a conjugating enzyme (E2) that receives ubiquitin from an E1; and an ubiquitin ligase (E3) that catalyzes the transfer of ubiquitin from the E2 active-site cysteine to a substrate (4). To date, 2 E1s and dozens of E2 enzymes have been identified in mammals, whereas the number of E3s is in the range of several hundreds (5-8). Many E3 enzymes contain a RING finger domain that interacts transiently with a cognate E2 to mediate polyubiquitination reactions (4, 9-12).Ubiquitination often occurs in the form of a polymer whereby the C-terminal Gly-76 in an ubiquitin moiety is linked to a lysine residue in another ubiquitin molecule. It had been presumed that polyubiquitin chains are formed by conjugating ubiquitin moieties, one at a time, first to a lysine residue in a substrate, and then to a lysine in the previously-attached ubiquitin molecule. This appears to be the case for some E2s (13-15). However, we and several other groups recently found that ubiquitin chains can also be preassembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its yeast homologue Ubc7p both in vitro and in vivo (16)(17)(18)(19). These ubiquitin-conjugating enzymes are associated with endoplasmic reticulum (ER) membranes to modify misfolded polypeptides that have been exported from the ER lumen in a proc...

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confidence: 65%

Section: Results Gp78 Contains 2 Oligomerization Sites One Of Which mentioning

confidence: 99%

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

115

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“…Although the activation of the intrinsic activity of a CRL is one mode of regulation by neddylation, it is not the only one. Indeed, CRL neddylation can block association with the CAND1 exchange factor and enhance association with the CSN complex, as described in detail below.In the on state, CRLs can be highly processive, giving rise to multiple catalytic cycles before substrate dissociation [70][71][72]. This feature has much to do with how a CRL-specific, ubiquitinchain-forming E2 associates with the cullin-RING E3 scaffold.…”

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confidence: 99%

Building and remodelling Cullin–RING E3 ubiquitin ligases

2013

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Cullin-RING E3 ubiquitin ligases (CRLs) control a plethora of biological pathways through targeted ubiquitylation of signalling proteins. These modular assemblies use substrate receptor modules to recruit specific targets. Recent efforts have focused on understanding the mechanisms that control the activity state of CRLs through dynamic alterations in CRL architecture. Central to these processes are cycles of cullin neddylation and deneddylation, as well as exchange of substrate receptor modules to re-sculpt the CRL landscape, thereby responding to the cellular requirements to turn over distinct proteins in different contexts. This review is focused on how CRLs are dynamically controlled with an emphasis on how c ullin neddylation cycles are integrated with receptor exchange. Keywords: cullin; ubiquitin; Nedd8; COP9 signalosome; E3 ligase; CAND1 EMBO reports (2013) 14, 1050-1061 published online 15 November 2013; doi:10.1038/embor.2013 See the Glossary for abbreviations used in this article. IntroductionThe proteome is constantly remodelled to suit the needs of the cell, through both synthesis and turnover. Protein turnover is controlled through two main systems: the ubiquitin-proteasome system (UPS) and lysosomal degradation system, including autophagy. Although selective autophagy can provide a means by which to control the abundance of particular organelles and even single proteins, it is best understood as a means to control bulk turnover of cellular components [1]. Conversely, the UPS is a particularly versatile and highly regulated system [2] that allows selective turnover of individual regulatory proteins, even when they are incorporated into higher-order multiprotein assemblies. On the one hand, the entire population of a given protein targeted by the UPS might be subject to rapid tagging with ubiquitin and degradation by the proteasome. On the other hand, the UPS might control the degradation of only a small pool of a particular target protein, for example the population of a protein that has been phosphorylated by an upstream pathway. Thus, the UPS functions in space and time to sculpt the proteome and to control the abundance of active or inactive forms of signalling complexes and cellular machines. As illustrated in this Review, the UPS machinery that controls turnover of a wide swathe of the p roteome is itself dynamically regulated through many mechanisms.The tagging of proteins with particular types of ubiquitin chains serves to mark them for proteasomal degradation. This process occurs through an E1 (ubiquitin-activating enzyme)-E2 (ubiquitinconjugating enzyme)-E3 (ubiquitin ligase) cascade [3][4][5][6][7]. E3 plays a central role in substrate targeting by binding directly to the substrate and presenting target lysine residues to receive ubiquitin from the E2, in the case of RING E3s, or from a ubiquitin-charged cysteine residue in HECT and RBR E3s [6,[8][9][10]. Cullin-RING E3 ubiquitin ligases (CRLs), which were first discovered almost two decades ago [11][12][13], are a superfamily of RING E3...

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“…Ubc1 cannot be recharged by E1 in this assay due to inactivation of E1 and free E2, and thus the assay measures a single turnover of E2, allowing estimates of Ubc1 catalytic rate and affinity for acceptor ubiquitin (3,18).…”

Section: Resultsmentioning

confidence: 99%

“…Cloning, Expression, and Purification of Proteins-To make 32 P-labeled K48R mutant ubiquitin for diubiquitin synthesis assays, GST-TEV-PKA-K48R-ubiquitin (a gift from Ray Deshaies (18)) was expressed in Escherichia coli, radioactively labeled, and purified as described (3). E2 constructs, including Ubc1-His 6 , Ubc1⌬UBA-His 6 , Ubc4-His 6 , and Ubc4-UBA-His 6 , were expressed in E. coli and purified as described (4).…”

Section: Methodsmentioning

confidence: 99%

An E2 Accessory Domain Increases Affinity for the Anaphase-promoting Complex and Ensures E2 Competition

Girard

Tenthorey

Morgan

2015

Journal of Biological Chemistry

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Mechanism of Lysine 48-Linked Ubiquitin-Chain Synthesis by the Cullin-RING Ubiquitin-Ligase Complex SCF-Cdc34

Cited by 254 publications

References 31 publications

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Building and remodelling Cullin–RING E3 ubiquitin ligases

An E2 Accessory Domain Increases Affinity for the Anaphase-promoting Complex and Ensures E2 Competition

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