Final Report Summary - AUTO-UBIQUITYLATION (Structural insight into auto-ubiquitylation as a regulatory mechanism for protein trafficking) We developed a synthetic biology approach that reconstitutes the entire ubiquitylation cascade in bacteria. Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and bio- physical characterisation of ubiquitylated proteins. We cir- cumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiqui- tylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterisation of several ubiquitylated proteins demon- strated the integrity, specificity and fidelity of the system, and revealed new biological findings. Recently, we also obtained crystal of ubiquitylated protein and were able to determined its structure. This is the first structure of ubiquitylated protein other than Ub itself, i.e. ubiquitylated-Ub.