Protein modification by a eukaryotic-like ubiquitin-related modifier in the hyperthermophilic archaeon Saccharolobus...

Although homologs of the eukaryotic Urm1 (ubiquitin-related modifier-1) have been characterized in Archaea, the bona fide substrates and roles of the archaeal Urm1 remain poorly understood. Here, we report a proteomic analysis of Urm1 modification in Saccharolobus islandicus using a highly efficient method, which involves the introduction of an H81R substitution into Urm1 encoded by the strain, treatment of the strain with the proteasome inhibitor bortezomib, and affinity enrichment of urmylated peptides with an anti-K-ε-Gly-Gly antibody following peptide fractionation. Extensive protein urmylation was observed, with a total of 783 Urm1 conjugation sites, mapped to 330 proteins, identified in the cell. Among the seven lysine residues in Urm1, six were sites of modification, of which K7 and K37 were preferentially modified. Treatment with the proteasome inhibitor bortezomib resulted in K37-linked chains being the sole major modification species, suggesting that K37 linkage served as a primary trigger of proteasomal degradation. The modified proteins were involved in a number of cellular processes, such as cell division, chromosomal organization, DNA replication, translation, proteasomal protein degradation, and sulfur relay. Protein urmylation was dynamic and influenced by growth conditions and stress treatments. Attempts to delete urm1 were unsuccessful, pointing to the essentiality of the gene. The knockdown of urm1 resulted in substantial growth delay, during which a drastic reduction in cellular concentration of cell division proteins (CdvB, CdvB1, CdvB2) occurred. Our results shed significant light on the landscape and potential roles of protein urmylation in Archaea.IMPORTANCEAlthough protein urmylation has been documented in Archaea for over a decade, the authentic substrates and functional roles of archaeal Urm1 remain largely unknown. In this study, we generated the largest Urm1 modification data set in Archaea through an efficient in vivo approach and investigated its physiological functions in Saccharolobus islandicus. Extensive protein urmylation was observed, with modified proteins implicated in key cellular processes such as cell division, chromosomal organization, translation, and proteasomal degradation. Our findings challenge the prevailing notion that Urm1 homologs modify only a limited number of substrates. Six out of seven lysine residues in Urm1 were modified, suggesting the presence of diverse Urm1 chain structures. These results provide cellular evidence supporting the hypothesis that eukaryotic Ub/Ubl systems have an archaeal origin. We also explored how various factors affect global protein urmylation and examined the impact of urm1 knockdown on cell growth.