The origins and evolution of ubiquitination sites

Protein ubiquitinationis central to the regulation of various pathways in eukaryotes. The process of ubiquitinationand its cellular outcome were investigated in hundreds of proteins to date. Despite this, the evolution of this regulatory mechanism has not yet been addressed comprehensively. Here, we quantify the rates of evolutionary changes of ubiquitinationand SUMOylation (Small Ubiquitin-like MOdifier) sites. We estimate the time at which they first appeared, and compare them to acetylation and phosphorylation sites and to unmodified residues. We observe that the various modification sites studied exhibit similar rates. Mammalian ubiquitinationsites are weakly more conserved than unmodified lysine residues, and a higher degree of relative conservation is observed when analyzing bona fide ubiquitinationsites. Various reasons can be proposed for the limited level of excess conservation of ubiquitination, including shifts in locations of the sites, the presence of alternative sites, and changes in the regulatory pathways. We observe that disappearance of sites may be compensated by the presence of a lysine residue in close proximity, which is significant when compared to evolutionary patterns of unmodified lysine residues, especially in disordered regions. This emphasizes the importance of analyzing a window in the vicinity of functional residues, as well as the capability of the ubiquitinationmachinery to ubiquitinateresidues in a certain region. Using prokaryotic orthologs of ubiquitinatedproteins, we study how ubiquitinationsites were formed, and observe that while sometimes sequence additions and rearrangements are involved, in many cases the ubiquitinationmachinery utilizes an already existing sequence without significantly changing it. Finally, we examine the evolution of ubiquitination, which is linked with other modifications, to infer how these complex regulatory modules have evolved. Our study gives initial insights into the formation of ubiquitinationsites, their degree of conservation in various species, and their co-evolution with other posttranslational modifications.