LAUSR

Significance The cell is a hive of information flow in which metabolite signals and proteins translocate across subcompartments. Peering into this submicroscopic world is hugely challenging. Using a strategy termed Localis-rex, we identify subcellular locale-specific sensors of a native reactive electrophilic metabolite. Surprisingly, several proteins sense electrophiles in locales where they do not canonically reside. One example is the nuclear protein, CDK9. In the cytosol, the electrophilic-metabolite–modified CDK9 has no negative function. However, following nuclear translocation, the electrophilic-metabolite–modified CDK9 downregulates the transcriptional activator, RNA-Polymerase-II. This exquisitely-nuanced signaling modality highlights the need to assign means, motive, and opportunity to proteins involved in reactive metabolite signaling. Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs—despite their direct relevance to covalent drug development—impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs. In a setup that contrasts nucleus- vs. cytoplasm-specific responsivity to reactive-metabolite modification (hydroxynonenylation), ∼40% of the top-enriched protein sensors investigated respond in compartments of nonprimary origin or where the canonical activity of the protein sensor is inoperative. CDK9—a primarily nuclear-localized kinase—was hydroxynonenylated only in the cytoplasm. Site-specific CDK9 hydroxynonenylation—which we identified in untreated cells—drives its nuclear translocation, downregulating RNA-polymerase-II activity, through a mechanism distinct from that of commonly used CDK9 inhibitors. Taken together, this work documents an unmet approach to quantitatively profile and decode localized and context-specific signaling/signal-propagation programs orchestrated by reactive covalent ligands.