Functional reconstitution of a tunable E3-dependent sumoylation pathway in Escherichia coli.
SUMO (small ubiquitin-related modifier) is a reversible post-translational protein modifier that alters the localization, activity, or stability of proteins to which it is attached. Many enzymes participate in regulated SUMO-conjugation and SUMO-deconjugation pathways. Hundreds of SUMO targets are currently known, with the majority being nuclear proteins. However, the dynamic and reversible nature of this modification and the large number of natively sumoylated proteins in eukaryotic proteomes makes molecular dissection of sumoylation in eukaryotic cells challenging. Here, we have reconstituted a complete mammalian SUMO-conjugation cascade in Escherichia coli cells that involves a functional SUMO E3 ligase, which effectively biases the sumoylation of both native and engineered substrate proteins. Our sumo-engineered E. coli cells have several advantages including efficient protein conjugation and physiologically relevant sumoylation patterns. Overall, this system provides a rapid and controllable platform for studying the enzymology of the entire sumoylation cascade directly in living cells.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America.
Sean P
SP
O'Brien
Matthew P
MP
DeLisa
D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D025842:SUMO-1 Protein / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism; D058207:Sumoylation / Q000235:genetics / Q000502:physiology*; D053719:Tandem Mass Spectrometry; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D017931:DNA Primers; C497796:SMAD4 protein, human; D025842:SUMO-1 Protein; D051901:Smad4 Protein; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases