Eukaryotic cells form biomolecular condensates to sense and adapt to their environment1,2. Poly(A)-binding protein (Pab1), a canonical stress granule marker3,4, condenses upon heat shock or starvation, promoting adaptation5. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogen-deuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1's condensation. We find that Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensate's interaction network. Our data yield a clear molecular picture of Pab1's stress-triggered condensation, which we term sequential activation, wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity. Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

Poly(A)-binding protein (Pab1 in yeast) is a canonical stress granule marker. Upon cellular stress including heat shock and starvation, Pab1 phase separates, demixing from the cytosol into foci in vivo. Similarly in vitro, the protein phase separates at heat shock temperatures (above 40 C) and at acidic pHs that occur during other stresses including starvation. Pab1 contains four highly charged RNA recognition motifs (RRMs), connected by linkers, followed by an intrinsically disordered proline-rich (P-) domain and a C-terminal peptide binding domain. We have shown that Pab1 demixing during stress is an adaptive response that can be modulated by hydrophobic mutations in its intrinsically disordered P-domain region (Riback et al (2017) Cell 168:1028. However, the role of Pab1's RRMs, which are necessary and sufficient for temperature demixing in vitro and in vivo, remain unclear. Here we further elucidate the molecular mechanism of Pab1's RRMs on the pH dependence of T demix , the demixing temperature. Since T demix drops rapidly as pH is lowered below physiological pH, we investigated the role of Pab1's 8 histidines, located in its RRMs, by substituting them with the consensus residues in the Pab1 family. Variants having multiple histidines replaced in a single RRM domain exhibit a slightly elevated T demix at all pH values. The variant missing all 8 histidines exhibits a greatly diminished pH sensitivity with its T demix remaining nearly constant, effectively abolishing the pH dependence of demixing within a physiological range. Overall, we find that T demix is proportional to net charge, whether positive or negative, regardless of the substituted positions. Additional factors in the demixing process will be discussed, including linker flexibility and stacking of the RRMs. Together, our studies address how cellular stress is transduced through Pab1's structure to promote its phase separation.

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