Step by step | Nature Reviews Molecular Cell Biology

Access through your institution Buy or subscribe Chain extension could occur by stepwise addition of ubiquitin monomers, by transfer of a preassembled chain from the enzymatic machinery to

the substrate (_en bloc_ transfer) or by a combination of the two mechanisms. Pierce _et al_. visualized ubiquitylation of a model substrate by the Cdc34–SCFCdc4 (E2–E3) complex in a

single-encounter reaction and found that ubiquitylation was complete in 30 seconds — too quick to discern the mechanism of chain extension. So, they measured the distribution of ubiquitin

chain lengths on Cdc34 and the number of ubiquitin transfer events from Cdc34 to the substrate, and tested the results in a theoretical model of the mechanisms of chain formation. The

results predicted that Cdc34–SCFCdc4 assembles ubiquitin chains on substrates primarily by sequential transfers of single ubiquitin molecules. This 'step by step'

polyubiquitylation mechanism was confirmed by data from single-encounter reactions on a quench flow apparatus, which allowed the authors to take measurements of the product distribution on a

millisecond timescale. Kleiger _et al_. studied how the speed of ubiquitylation is achieved. They measured the dynamics of the Cdc34–SCFCdc4 interaction and found that the two enzymes bind

and dissociate rapidly. Based on normal rates of protein association, the rates of E2 recruitment and dissociation for an E2–E3 complex with submicromolar affinity would be too slow to

sustain the maximal rate of synthesis of a ubiquitin chain reported by Pierce _et al_. (∼5 molecules per second). Substrates would dissociate before chain synthesis could be completed.

However, rapid association kinetics have been previously observed for interacting proteins that align through electrostatic interactions, and Cdc34 has an acidic tail that might behave the

same. Indeed, deletion of the acidic tail of Cdc34 caused defects in SCFCdc4 binding, suggesting that electrostatic interactions might be important for complex formation. This is a preview

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ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support REFERENCES ORIGINAL RESEARCH PAPERS * Kleiger, G. et al. Rapid E2-E3

assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. _Cell_ 139, 957–968 (2009) Article  CAS  Google Scholar  * Pierce, N. W. et al.

Detection of sequential polyubiquitylation on a millisecond timescale. _Nature_ 3 Dec 2009 (doi:10.1038/nature08595) Article  CAS  Google Scholar  FURTHER READING * Ye, Y. & Rape, M.

Building ubiquitin chains: E2 enzymes at work. _Nature Rev. Mol. Cell Biol._ 10, 755–764 (2009) Article  CAS  Google Scholar  Download references Authors * Francesca Cesari View author

publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cesari, F. Step by step. _Nat

Rev Mol Cell Biol_ 11, 7 (2010). https://doi.org/10.1038/nrm2824 Download citation * Published: 09 December 2009 * Issue Date: January 2010 * DOI: https://doi.org/10.1038/nrm2824 SHARE THIS

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