Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Abstract

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 biophysical characterization of ubiquitylated proteins. We circumvented 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 monoubiquitylation 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. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

Spatiotemporal Regulation of the Ubiquitinated Cargo-binding Activity of Rabex-5 in the Endocytic Pathway

“…Therefore, we proved that the MIU domain is not only involved directly in the trafficking of ubiquitinated cargo but also plays a key role in the coupled monoubiquitination of Rabex-5 to inactivate its ubiquitinated cargo-binding activity. However, our results differ from a previous study by Keren-Kaplan et al (42), which reported that ubiquitination occurs on the Vps9-GEF domain and that it does not affect the GEF activity in vitro. This could be attributed to the different types of UBDs present in human Rabex-5 and yeast Vps9 proteins.…”

A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

“…There have also been advances in assays to assess proteasomal inhibition [11], FRET-based sensors to quantify single cell proteasome activity [12], and substrates to identify the importance of protein folding on proteasomal targeting and degradation kinetics [13]. Further, tools exist that can distinguish between different ubiquitin species (free, mono-, or polyubiquitin chains) in the cytosol [14] and perform biochemical and biophysical characterization of E3 ligase kinetics independent of DUBs in a synthetic ubiquitination cascade in bacteria [15]. Additionally, novel proteomics-based approaches have been developed using mass spectrometry and stable isotope labeling by amino acids in cell culture (SILAC) to perform high throughput identification of E3 ligase substrates [16], [17].…”

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

“…To overcome the inhibitor effects of DUBs, one group sought to reconstitute the E1-E2-E3 enzymatic cascade in bacteria (49). Co-expression of affinity-tagged substrates and ubiquitin resulted in purified proteins that were used to study E3 autoubiquitination and map ubiquitination sites on the E3 ligase Mindbomb.…”