Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

Significance RAS proteins, critical regulators of cell growth and differentiation, are the most frequently mutated oncogenes in humans. RAS functions as dimers/coclusters on cell membranes. We developed an improved split luciferase complementation assay coupled to a powerful genetic system to show that colocalization within the same membrane domain enables formation of RAS dimers/coclusters with itself and other membrane-associated proteins. Membrane association-facilitated interactions (MAFIs) are not sufficient for RBD-mediated Ras inhibition, which additionally requires high-affinity domain-mediated interactions. Notably, we show that MAFI augments the impact of domain-mediated interactions to elicit autophagy/lysosome-mediated elimination of nonfunctional RAS complexes. This broadly applicable strategy enables discovery of low-affinity protein interactions mediated by membrane tethering and analysis of their impact on biologic function.