Ribosome heterogeneity provides a translational barrier against protein misfolding disease in Saccharomyces cerevisiae.
The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor Sup35p (eRF3). Among 79 ribosomal proteins in Saccharomyces cerevisiae, through a genetic screen for novel anti-prion factors, we identified RPL24B, a 60S ribosomal subunit protein, as a key determinant of prion generation. Our findings revealed that the functional distinction between the ribosomal paralogs RPL24A and RPL24B is the primary driver of ribosomal heterogeneity-based anti-prion generation. Heterogeneity establishes a critical genetic checkpoint that strictly regulates the emergence and heritable diversity of prions and their variants. We demonstrate that deleting RPL24B (rpl24bΔ) significantly increased the frequency of [PSI+] generation compared to the wild-type strain. Crucially, the resulting [PSI+] prions in the rpl24bΔ mutants exhibited distinct heritable diversity, characterized by high thermostability and exceptionally high propagon numbers. This anti-prion activity is uniquely specific to the RPL24B paralog, because deletion of its highly homologous counterpart, RPL24A, does not reproduce the effects observed in rpl24bΔ. Mechanistically, we identified valine 138 (V138) in Rpl24b as the major molecular determinant of its anti-prion generation activity. Loss of Rpl24b perturbs the cellular protein quality control landscape, leading to high accumulation of misfolded protein aggregates. This work suggests that RPL24B functions as a specialized retainer of genetic robustness, providing a translational barrier against protein misfolding diseases by filtering out robust amyloid variants.