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. 2021 Jun 18;10:e60660. doi: 10.7554/eLife.60660

Figure 5. Linear ubiquitin chain assembly complex (LUBAC) assembles heterotypic poly-ubiquitin chains containing M1 and ester bond linkages at T12 and T55.

(A) Treatment of LUBAC-assembled heterotypic poly-ubiquitin chains with hydroxylamine. (B) Hydroxylamine treatment of ubiquitin polymers assembled by LUBAC using N-terminally blocked ubiquitin. (C) MS/MS spectra of ubiquitin polymerized at T55 (top) and T12 (bottom). Poly-ubiquitin chains assembled by LUBAC were separated by SDS-PAGE, bands were cut from the gel and subjected to mass spectrometry analysis. (D) Positions of Thr12 and Thr55 on structure of ubiquitin (PDB:1UBI). (E) Assembly of ubiquitin chains by LUBAC using different ubiquitin Thr to Val point mutants as substrates. All experiments were performed in triplicate representative results are shown.

Figure 5.

Figure 5—figure supplement 1. Cezanne, vOTU, and hydroxylamine can cleave oxyester bonds in linear ubiquitin chain assembly complex (LUBAC)-assembled heterotypic ubiquitin chains.

Figure 5—figure supplement 1.

(A) Combined OTULIN and hydroxylamine treatment of LUBAC-assembled poly-ubiquitin chains. (B) Cezanne and vOTU ubiquitin chain restriction (UbiCRest) analysis of ubiquitin polymers assembled by LUBAC using N-terminally blocked ubiquitin.