Metabolically regulated proteasome supramolecular organization in situ.

Abstract

Many proteins localize in membraneless organelles. However, understanding the steps along membraneless organelle formation-and the structural impact on granule constituents-has been hindered by limited resolution of intracellular data. We address these challenges through in situ cryo-electron tomography (cryo-ET) along with formation of yeast proteasome storage granules (PSGs). During the transition from proliferation to quiescence, doubly capped 26S proteasomes arrested in an inactive state arrange into ∼7.5 MDa trimeric units, dispersed in the nucleoplasm and congregated along the nuclear envelope near the nuclear pore. 9-Å-resolution cryo-ET structures reveal that cytoplasmic PSGs formed in various energy-limiting conditions are paracrystalline arrays of bundled fibers, assembled from stacking of proteasome trimers. The paracrystalline arrangement maintains a pool of fully assembled inactive 26S proteasomes that are released in energy-rich conditions. Overall, our data reveal structural steps along the assembly of an intracellular membraneless organelle in situ and quinary structure formation controlling a major eukaryotic regulatory machine.