Fig. 5: Application of SUE1 to defined multisite ubiquitinated proteins and site-specific NEDD8-modified proteins.

a Schematic representation of SUE1-mediated multisite ubiquitination, in which protein of interesting (POI) bears multiple (here two) SUE1 tags (KEGYEE) at customized sites. Generally, 1 μM Uba1, 20 μM UBE2E1, 8 μM substrate containing multiple SUE1 tags, 40 μM monoUb or Ub chain were mixed and reacted in the reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂ and 10 mM ATP) at 37 °C for 2 h. b SDS‒PAGE analysis of SUE1 mediated dual-site monoubiquitination at K43 and K96 of α-synuclein (α-Syn). c Schematic representation of the SUE1 strategy in combination with the LACE strategy for multi-site ubiquitinated proteins with different Ub chain linkages, in which protein of interesting (POI) bears the SUE1 tag and LACE tag at customized sites. d SDS–PAGE analysis of SUE1 and LACE co-mediated dual-site ubiquitination of α-synuclein (α-Syn) and K48-linked diUb modification at K43 by SUE1 strategy and monoubiquitination at K96 by LACE strategy. In general, 1 μM Uba1, 40 μM UBE2E1, 16 μM substrate containing one SUE1 tag and one LACE tag and 16 μM Ub chain (or monoUb) were mixed and reacted in reaction buffer to achieve almost complete ubiquitination of substrate, and then 1 μM chimera E1 V4.5, 40 μM UBC9 K14R and 160 μM another Ub chain (or monoUb) were added to the reaction buffer at 37 °C for another 2 h. e Schematic representation of Uba1/UBE2E1-mediated site-specific neddylation, in which protein of interesting (POI) bears the SUE1 tag (KEGYEE) at the customized site. The reaction conditions were the same as for mono-ubiquitination, except NEDD8 (N8) was used to replace monoUb. f SDS‒PAGE analysis of SUE1-Mediated NEDD8-modification of EGFP. g deconvoluted ESI-MS of the purified neddylated EGFP (EGFP-N8). Partially EGFP was gluconoylated on His tag (+178 Da) during expression in E. coli. Gel images shown in (b), (d) and (f) are representative of independent biological replicates (n = 2). Source data are provided as a Source Data file.