DOI:10.1038/nrm2232

Ubiquitylation requires the subse-quent actions of three enzymes: an activating E1, a conjugating E2 and an E3 ligase. Hoeller et al. now show that monoubiquitylation of ubiquitin-binding domain (UBD)-containing substrates also occurs in the absence of E3 ligase activity.

It is well documented that pro-teins that can bind ubiquitin (Ub) non-covalently are often monoubiq-uitylated themselves. It is thought that this phenomenon of ‘coupled monoubiquitylation’ may lead to an intramolecular cis interaction of Ub with the UBD, which prevents

trans interactions of the UBD with other ubiquitylated proteins. Hoeller et al. hypothesized that monoubiq-uitylation of UBD-containing proteins might be achieved without the need for an E3 ligase. Indeed, in vitro ubiquitylation reactions with a panel of E2 enzymes in the presence of Ub, E1 and ATP resulted in the monoubiquitylation of a selection of UBD-containing substrates, albeit with various efficiencies and speci-ficities. However, when the authors co-expressed a UBD-containing substrate together with a UBD mutant that was not ubiquitylated, they found that UBD-containing proteins were unable to promote the transfer of Ub to another substrate in trans.

Mutations of various UBDs that disrupted the binding to Ub abolished E3-independent monoubiquitylation in vitro. Similarly, a Ub mutant that was unable to interact with a UBD was not conjugated to the UBD-containing substrate. The apparent requirement of a functional Ub–UBD interface for E3-independent mono-ubiquitylation was confirmed by fluo-rescence resonance energy transfer (FRET) studies in vivo. Co-expression of a substrate and an E2 mutant that was unable to bind to E3 ligases (both tagged with fluorescent markers) led to a similar FRET signal as when wild-type E2 was co-expressed with the substrate. By contrast, the FRET signal was extremely low when using either a mutant E2 or a mutant

substrate that were both unable to bind Ub. The FRET studies therefore show a direct interaction between substrate and Ub-loaded E2.

Finally, Hoeller et al. tested whether E3-independent mono-ubiquitylation also occurs in living cells. The loss of E3 ligases did not affect monoubiquitylation of several UBD-containing proteins. However, knockdown of the expression of the two most potent E2 enzymes for sev-eral substrates resulted in a marked decrease in monoubiquitylation of these substrates. This effect could be rescued by transfecting the cells with knockdown-resistant wild-type E2 enzyme or an E2 mutant that was unable to bind E3 ligase.

So, there appear to be multiple ways by which UBD-containing proteins can be modified. But what is the physiological significance of E3-independent monoubiquitylation? The authors propose that it might be responsible for maintaining the homeostasis of monoubiquitylated and non-ubiquitylated forms of UBD-containing proteins in the cell. Structural studies in the authors’ laboratory are already under way that will hopefully shed light on the mechanism of E3-independent monoubiquitylation.

Arianne Heinrichs

ORIGINAL RESEARCH PAPER Hoeller, D. et al. E3-independent monoubiquitination of ubiquitin-binding proteins. Mol. Cell 26, 891–898 (2007)FURTHER READING Hicke, L. et al. Ubiquitin-binding domains. Nature Rev. Mol. Cell Biol. 6, 610–621 (2005) l Welchman, R. L. et al. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nature Rev. Mol. Cell Biol. 6, 599–609 (2005)WEB SITEIvan Dikic’s laboratory: http://www.zbc.kgu.de/biochemie2/molsig/home_molsig.html

P O S T- T R A N S L AT I O N A L M O D I F I C AT I O N

It doesn’t take three

… that mono-ubiquit ylation of UBD-containing proteins might be achieved without the need for an E3 ligase.

R E S E A R C H H I G H L I G H T S

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | AUGUST 2007

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