Data underlying all figures.

Many proteins contain intrinsically disordered regions (IDRs) that are essential for their function but do not adopt a stable structure; instead, they exist as an ensemble of conformations. Because these regions lack fixed structural constraints, traditional structure-based and alignment-based approaches are often ineffective for studying their sequence–function relationships. Here, we present an approach that combines molecular evolution with genetic complementation to extract functional sequence features of an IDR. We use the budding yeast RNA-binding protein Rim4 as a model system. Rim4 is required for sporulation, and its IDR facilitates its dual role as a translational activator and repressor. Notably, Rim4’s IDR supports assembly into an SDS-resistant amyloid-like form, which is required for its repressor function. We demonstrate that the Rim4 IDR is functionally conserved across orthologous sequences spanning more than 400 million years of evolution, despite extensive sequence divergence. Our results suggest that noncomplementing Rim4 IDRs generally evolve toward higher hydrophobicity, and that reducing hydrophobicity can refunctionalize a nonfunctional IDR sequence that diverged over 200 million years ago. In the refunctionalized IDR, the activator function is restored, whereas assembly into an amyloid-like form remains uncomplemented. Overall, our findings add to evidence that IDRs can perform multiple functions, with each role optimized by distinct biochemical properties, and that evolutionary pressure favoring one function may drive the IDR toward biochemical characteristics that compromise its other functions.