Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates

While Ras is known to dynamically shuttle around the cell, the activity, mechanism of activation, and function of non-plasma membrane-localized Ras is unclear due to lack of suitable tools. To address these questions, we used the Latching Orthogonal Cage-Key pRotein (LOCKR) switch platform to generate first-in-class intracellular sensors for endogenous Ras activity (Ras-LOCKR-S) and signaling-dependent proximity labelers (Ras-LOCKR-PL). Localizing these tools to endomembranes and oncogenic condensates, we detected local Ras activity and identified upstream Ras effectors (guanine exchange factors and SAM68) responsible for signaling in these locations. We found further that Major Vault Protein drives RasG12C inhibitor resistance by enhancing signaling at the golgi and altering mitochondrial metabolism during prolonged drug treatment. Together, these results highlight the importance of non-plasma membrane Ras signaling (endomembranes and condensates), and our new sensors should accelerate the discovery of new therapeutic targets. ### Competing Interest Statement J.Z.Z., D.J.M., and D.B. are co-inventors in a provisional patent application (application number 63/380,884 submitted by the University of Washington) covering the biosensors described in this manuscript.