The conjugation of ubiquitin to target molecules involves several enzymatic steps. Little is known about the specificity of ubiquitination. How E3 ligases select their substrate and which lysines are targeted for ubiquitin conjugation is largely an enigma. The object of this study is to identify preferred ubiquitination sites. Genetic approaches to study this question have proven difficult, because of the redundancy of ligases and the lack of strictly required motifs. However, a better understanding of acceptor site selection could help to predict ubiquitination sites and clarify yet unsolved structure-function relationships of the transfer reaction.