Redistribution of proteasomes through diffusion and cytoskeleton-dependent mechanisms upon stress induced by protein aggregates

Proteasomes are abundant molecular machines distributed throughout the eukaryotic cell to facilitate protein degradation. We previously showed that proteasomes can re-organize and assemble into foci bodies in response to proteotoxic stress1, which we termed transient aggregate-associated droplets (TAADs)2. Here, we use single-molecule localization microscopy to quantify re-organization of mammalian proteasomes in different subcellular regions during proteotoxic stress to engage with invading protein aggregates. We determine that [~]60% of 20S proteasomes are capped by at least one 19S particle, and this ratio remains constant during stress. Using single-particle tracking, we show that cells confine global proteasomal movement when experiencing proteotoxic stress via a cytoskeleton-dependent mechanism. Similar limitations on proteasome movement are also observed upon membrane depolarization and repolarization events by patch clamp, which directly induce proteasomes to move away or towards the plasma membrane, respectively. Together, our experiments reveal distinct modes of proteasome motion dependent on cellular requirements, and propose that proteasome transport is restricted upon aggregate invasion to facilitate their subsequent degradation.