Detection of sequential polyubiquitylation on a millisecond timescale

Abstract

The pathway by which ubiquitin chains are generated on substrate via a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCF Cdc4 and SCF β-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.Attachment of a polyubiquitin chain with at least four ubiquitins linked together through their lysine 48 residue (Lys48) targets proteins to the proteasome for degradation.1 A cascade of three enzymes carries out the synthesis of polyubiquitin chains: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3).2 RING (really interesting new gene) E3s catalyze the direct transfer of ubiquitin from an E2 to a lysine on a target protein.3 SCF Cdc4 is the founding member of the largest family of E3s -the cullin-RING ubiquitin ligases (CRLs) that may comprise the majority of all human ubiquitin ligases.3 Thus, unraveling the mechanism of SCF will have broad functional ramifications for the preponderance of human E3s.Different pathways for ubiquitin chain assembly by RING E3s have been envisioned based on indirect evidence. On the one hand, Cdc34-SCF ubiquitylates substrates bearing a single Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms 3 Corresponding author. deshaies@caltech.edu. * These authors contributed equally to this work. Author ContributionsN.W.P. performed all computational modeling and experiments except G.K. performed the mass spec experiments in Fig. 1g. N.W.P., R.J.D., and S.O.S. conceived the experiments. N.W.P. and R.J.D. wrote the manuscript with editorial input from the other authors. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript ubiquitin significantly faster than non-ubiquitylated substrates,4,5 suggesting that it processively builds polyubiquitin chains on substrates with an initial slow transfer of ubiquitin followed by rapid elongation into a Lys48-linked polyubiquitin chain. On the other hand, the E2 Ube2g2, a close relative of Cdc34, collaborates with the E3 gp78 to build a polyubiquitin chain on its a...

Building and remodelling Cullin–RING E3 ubiquitin ligases

“…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.…”

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

“…We next compared the kinetics of San1 peptide ubiquitylation with the kinetics of a reaction catalyzed by the SCF ubiquitin ligase as the rates of ubiquitin transfer to SCF-bound substrates are among the fastest in the literature (54). Singleturnover ubiquitylation reactions were assembled with either yeast Cdc34 or Ubc1 and San1 or with SCF in combination with both UbcH5c and human Cdc34 (note that SCF has been shown to function synergistically with these E2s; Ref.…”

“…Yeast WT and ⌬190 Cdc34 were expressed and purified as previously described (51,54). Yeast Ubc1 was expressed and purified essentially as previously described (46) with the following modifications.…”

“…Thus, fast association also enables a rapid rate of Cdc34 dissociation from SCF without compromising high affinity binding. Rapid dynamics between Cdc34 and SCF promotes processive ubiquitin transfer (54), increasing the probability that a substrate-E3 encounter will result in substrate ubiquitylation and degradation.…”

A SnapShot of Ubiquitin Chain Elongation

“…Quench-flow experiments have revealed that SCF/Cdc34 catalyzes ubiquitination in a progressive manner, with the addition of one Ub at a time (30). SCF appears to engage Cdc34 during the elongation reaction via a two-step mechanism.…”