Synthetic biology approach to reconstituting the 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 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.
Pubmed ID: 22081111 RIS Download
Adaptor Proteins, Vesicular Transport | Affinity Labels | Cloning, Molecular | Escherichia coli | Fluorescence Resonance Energy Transfer | Genetic Vectors | Guanine Nucleotide Exchange Factors | Guanosine Diphosphate | Plant Proteins | Proteasome Endopeptidase Complex | Protein Processing, Post-Translational | Recombinant Fusion Proteins | Saccharomyces cerevisiae Proteins | Substrate Specificity | Synthetic Biology | Ubiquitin | Ubiquitin-Activating Enzymes | Ubiquitin-Conjugating Enzymes | Ubiquitin-Protein Ligase Complexes | Ubiquitin-Protein Ligases | Ubiquitination | Vesicular Transport Proteins | Zebrafish Proteins