Figure 3: Production of covalent ATP-dependent Urm1 conjugates by the ELSA/Uba4p/ThiF E1-like enzyme.

(a) Auto-urmylation of the ELSA/Uba4p/ThiF enzyme. 30 μg N-terminally His-tagged Urm1 protein and 90 μg ELSA/Uba4p/ThiF enzyme were incubated together at 70 °C for 1 h in either the presence or absence of 2.5 mM ATP, and retrieved by Ni-NTA agarose pulldown. Left; Coomassie stained gel of the pull-down. Right; western blot of a duplicate gel, probed with an anti-His antibody. Lanes 1 and 4: Urm1 only controls, without or with ATP, respectively; lanes 2 and 5: untagged ELSA/Uba4p/ThiF only controls, without or with ATP, respectively; lanes 3 and 6: Urm1 plus ELSA/Uba4p/ThiF incubations, without or with ATP, respectively. (b) Urmylation of the Saci0666 thermosome subunit. 30 μg N-terminally His-tagged Urm1 protein plus 15 μg ELSA/Uba4p/ThiF enzyme and 75 μg C-terminally His-tagged Saci0666 were incubated together and pulled-down as described in (a). Lane 1: reaction in the presence of 2.5 mM ATP; lane 2: no ATP control. Left; Coomassie stained gel of the pull-down. Middle and Right; western blot of duplicate gels, probed with an anti-His, or anti-Urm1 antibodies, respectively. (c) Example MS/MS spectra of the diglycine modified peptides from the in vitro urmylation assay (Saci0671 (FBP) and Saci0669 (Urm1)) and in vivo Urm1 overexpression (Saci0051 [Rad50] and Saci0656 [PAN]). m/z values of the precursor ions are shown in the top left of each panel. (2+) or (3+) indicates doubly or triply charged precursor ions, respectively. Spectra show the annotated peaks that are due to C-terminal y (coloured red) and N-terminal b (coloured blue) fragment ions. In each case, the m/z values of the precursor ions and the m/z values of the fragment ions are consistent with diglycine modified lysine residues. The amino-acid sequence of the chymotrypsin-generated peptide, including the di-glycine modified lysine, is shown within the grey box in each example. Other examples are shown in Supplementary Fig. 5.