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The pathway by which ubiquitin chains are generated on substrate through 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) E3 enzymes SCF^sup Cdc4^ and SCF^sup 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 (Lys 48) targets proteins to the proteasome for degradation1. A cascade of three enzymes performs the synthesis of polyubiquitin chains: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3)2. RING E3 enzymes catalyse the direct transfer of ubiquitin from an E2 to a lysine on a target protein3. SCF^sup Cdc4^ is the founding member of the largest family of E3 enzymes-the cullin-RING ubiquitin ligases that may comprise most of all human ubiquitin ligases3. Thus, unravelling the mechanism of SCF will have broad functional ramifications for the preponderance of human E3 enzymes.
Different pathways for ubiquitin chain assembly by RING E3 enzymes have been envisioned based on indirect evidence. On the one hand, Cdc34-SCF ubiquitylates substrates bearing a single ubiquitin significantly faster than non-ubiquitylated substrates4,5, suggesting that it processively builds polyubiquitin chains on substrates with an initial slow transfer of ubiquitin followed by rapid elongation into a Lys-48-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 active site cysteine that can be transferred en bloc to substrate6,7. Various permutations of the en bloc mechanism have been entertained, in which the chain is built either from proximal to distal end or...