Rapid depletion of target proteins allows identification of coincident physiological responses

Abstract

Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involvemodifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using smallmolecules. For each target, substantial optimizationmay be required to achieve a prac-tical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough tomanifest a physiological response. Here, we describe a simplified targeted degradation systemthat rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological responsemanifests that is related to the function of the target. Using ribo-somal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of severalmRNAs, even before appreciable cell division has occurred. © 2012, American Society for Microbiology.