Systematic identification of pH-sensing amyloid core motifs reveals a widespread mechanism for reversible protein assembly upon stress

<jats:title>Abstract</jats:title> <jats:p> Unlike irreversible pathological amyloids, reversible fibrils can be regulated via pH-sensing core motifs, characterized by amyloid properties unleashed upon stress-induced protonation of critical residues. Here, we combined bioinformatic predictions and an <jats:italic>in vitro</jats:italic> validation pipeline to search for pH-responsive, reversible amyloid core peptides in yeast and human proteomes. This approach uncovered biophysical properties distinguishing pH-sensing and constitutive amyloid cores and established reliable criteria to identify novel reversible assemblies based on sequence data. Selected full-length candidate proteins with evolutionarily conserved pH-sensing motifs were analyzed in <jats:italic>Saccharomyces cerevisiae</jats:italic> using fluorescence microscopy and SDS-resistance assays, revealing multiple proteins forming reversible assemblies in stationary phase. We found that protonation of a specific histidine in the amyloid core motif of the asparagine synthase Asn1 is necessary and sufficient for assembling catalytically inactive, reversible structures called cytoophidia. Interestingly, mutant cells that fail to assemble Asn1-cytoophidia show defects to recover from stationary phase, demonstrating functional relevance of pH-sensing amyloid core motifs <jats:italic>in vivo</jats:italic> . Taken together, we uncovered a widespread and conserved pH-sensing mechanism that regulates the reversible assembly and function of structurally diverse fibrils upon stress. </jats:p>