Post-translational regulation of proteins using a light-controllable split-protease system

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Karl Gerhardt, Jeffrey Tabor

Rice University, United States

Proteolytic cleavage is a strategy utilized by both prokaryotic and eukaryotic cells in order to rapidly initiate a wide range of cellular processes. These include protein activation or inactivation by removal of inhibitory or catalytic domains, mediation of protein degradation by revealing stabilizing or destabilizing residues, and initiation of protein translocation by removal of peptide signal sequences or exposure of latent ones. By regulating such a diverse array of cellular processes, proteolytic cleavage presents itself as an excellent candidate for synthetic control. Here, we demonstrate in Dictyostelium discoideum a novel light-inducible protease system which has been constructed by fusing split halves of the NIa tobacco etch virus protease (TEVP) to the light dimerizing proteins phytochrome B (phyB) and PIF6. By coupling the proteolytic output of split-TEVP to the light input dependent activity of phyB and PIF6 we show that target proteins of interest can be modified to enter the N-end rule pathway for rapid protein degradation in a light-controllable manner. Finally, we are working to take full advantage of the precise spatiotemporal nature of light to control the numerous other forms of post-translational regulation which can be mediated by proteolytic cleavage. We envision this system being a general tool for making post-translational cellular processes easier to engineer and study by making it possible to control and perturb these processes on the same millisecond and sub-micron length scale that cells do.