Data on phase separation of Sup35p protein in Vivo

When yeast are energy depleted, Sup35 prion protein phase separates into condensates with very different properties than the phase separated Sup35 liquid droplets that form in vitro at low pH characterized by the Alberti laboratory (Franzmann et al. Science 2018). In contrast to Sup35 liquid droplets, which is driven by the prion domain of Sup35 and does not require the C-terminal domain, a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo. Phase separation of Sup35 in vivo requires the C-terminal domain, which binds to Sup45 to form the translation termination complex. The phase separated Sup35 not only colocalizes in vivo with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since treating the yeast with cycloheximide, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, the liquid droplets of Sup35 that form in vivo do not reversibly disassemble when the intracellular pH is increased unlike the purified Sup35 in vitro. Taken together, these results suggest that, in energy-depleted cells, Sup35 is incorporated into supramolecular assemblies whose properties differ from those of Sup35 liquid droplets that form in vitro.