E3-independent ubiquitination of AtMAPR/MSBP1

AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3 and AtMAPR5/MSBP1 were identified using LC–MS/MS. Representative MS/MS spectra showing the signature peptide Gly–Gly tag on Lys residue of ubiquitinated-AtMAPR3. [Display omitted] ► First report of the E3-independent ubiquitination of a plant-derived protein. ► Multiple E2s have the ability to ubiquitinate AtMAPR2. ► This ubiquitination reaction was dominated by mono-ubiquitination at multiple sites. AtMAPR5/MSBP1 and its homologs can be ubiquitinated in the absence of E3 ligase in in vitro ubiquitination assays. Ubiquitinated AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 were identified using LC–MS/MS. Analysis of trypsin-released signature peptides showed that this E3-independent ubiquitination of AtMAPR3, AtMAPR5/MSBP1, and AtMAPR2 was dominated by mono-ubiquitination at multiple sites. Unlike AtUBC8-type E2s, AtUBC36 was not able to transfer ubiquitin to AtMAPR2. The truncated mutants AtMAPR2Δ1–10, AtMAPR2Δ1–30, and AtMAPR2_1–73 could also be ubiquitinated. The presence of a ubiquitin-binding domain (UBD) allows proteins to be ubiquitinated independently of E3 ligases. However, AtMAPRs do not contain any known UBD. In vitro ubiquitination of AtMAPR2 observed in this study will be further studied in biochemical and physiological aspects.