Cells have evolved quality control mechanisms that operate under normal growth conditions and during stress to maintain protein homeostasis (proteostasis) and prevent the formation of potentially toxic aggregates. Research in recent decades has identified complex quality control systems in the cytoplasm that mediate protein folding, prevent misfolding, and cooperate in protein degradation with the proteasome and autophagy pathways. Compartment-specific proteostasis networks and stress response pathways have also been described for the endoplasmic reticulum and mitochondria. In contrast, relatively little is known about protein quality control in the nucleus.

Proteins enter the nucleus in a folded state, so chaperone machinery specific for de novo folding is not required. However, the nuclear proteome is rich in stress-sensitive, metastable proteins, which suggests that effective protein quality control mechanisms are in place to ensure conformational maintenance. The nucleus contains several non–membrane-bound subcompartments. The largest of these is the nucleolus, the site of ribosome biogenesis. During stress, Hsp70 and other molecular chaperones accumulate in the nucleolus, presumably to protect unassembled ribosomal proteins against aggregation. The nucleolus consists of liquid-like phases or domains that have differential surface tension and do not intermix. The outermost of these, the granular component (GC), is rich in negatively charged proteins such as nucleophosmin and nucleolin, which, combined with RNA, can undergo phase separation into liquid droplets in vitro, as shown for nucleophosmin.

Nuclear protein aggregates have been observed in various neurodegenerative disorders such as amyotrophic lateral sclerosis and Huntington’s disease, but protein quality control in the nucleus is not well understood. Here, we used a combination of fluorescence imaging, biochemical analyses, and proteomics to investigate the fate of stress-denatured and aberrant proteins in the nucleus, focusing specifically on the role of the nucleolus and its phase-separated nature in protein quality control.

Upon heat stress, misfolded nuclear proteins entered the liquid-like GC phase of the nucleolus, where they associated with proteins including nucleophosmin and adopted a state of low mobility. As a consequence, a fraction of nucleophosmin and nucleolin also converted to a less dynamic state. Storage in the GC phase effectively prevented the irreversible aggregation of misfolded protein species, allowing their extraction and refolding upon recovery from stress in a Hsp70-dependent manner. We identified ~200 different proteins that reversibly partitioned upon stress into the immobile substate of the GC, entering either from the nucleoplasm or from within the nucleolus. Disruption of the GC phase resulted in the formation of stable aggregates of stress-denatured proteins in the nucleoplasm, which exerted toxic effects by sequestering bystander proteins. Notably, the capacity of the nucleolus to store misfolded proteins proved to be limited. Prolonged stress or the uptake of aberrant proteins associated with neurodegenerative diseases led to a transition of the GC phase from a liquid-like to a solid state, with loss of reversibility and nucleolar dysfunction.

The liquid-like GC phase of the nucleolus functions as a non–membrane-bound protein quality control compartment. It is characterized by a remarkable chaperone-like capacity to temporarily store misfolded proteins, preventing their irreversible aggregation and maintaining them as competent for Hsp70-assisted refolding …