Research Article

The nucleolus functions as a phase-separated protein quality control compartment

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Science  26 Jul 2019:
Vol. 365, Issue 6451, pp. 342-347
DOI: 10.1126/science.aaw9157

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Phasing-in quality control in the nucleus

The fundamental process of protein quality control in the nucleus is not well understood. The nucleus contains several non–membrane-bound subcompartments forming liquid-like condensates. The largest of these is the nucleolus, the site of ribosome biogenesis. Frottin et al. found that metastable nuclear proteins that misfold upon heat stress enter the nucleolus. In the nucleolus, they avoid irreversible aggregation and remain competent for heat shock protein 70–dependent refolding upon recovery from stress. Prolonged stress or the uptake of proteins associated with neurodegenerative diseases prevented this reversibility. Thus, the properties of a phase-separated compartment can assist in protein quality control.

Science, this issue p. 342

Structured Abstract

INTRODUCTION

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.

RATIONALE

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.

RESULTS

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.

CONCLUSION

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. Nucleoplasmic proteins exit the nucleolus upon refolding, and nucleolar proteins resume their functional state. Our findings provide an example of how the properties of a non–membrane-bound, phase-separated compartment can be used in protein quality control, a fundamental biological function.

Inserting misfolded proteins into the nucleolus prevents irreversible aggregation.

Upon cell stress, misfolded proteins enter the GC phase of the nucleolus to be stored in a state competent for Hsp70-dependent refolding during recovery. Potentially toxic, irreversible aggregates form when transfer into the nucleolus is prevented. A 3D-rendered high-resolution image of the nucleolus is shown: GC, granular component (red); DFC, dense fibrillar component (white); FC, fibrillar center (cyan).

Abstract

The nuclear proteome is rich in stress-sensitive proteins, which suggests that effective protein quality control mechanisms are in place to ensure conformational maintenance. We investigated the role of the nucleolus in this process. In mammalian tissue culture cells under stress conditions, misfolded proteins entered the granular component (GC) phase of the nucleolus. Transient associations with nucleolar proteins such as NPM1 conferred low mobility to misfolded proteins within the liquid-like GC phase, avoiding irreversible aggregation. Refolding and extraction of proteins from the nucleolus during recovery from stress was Hsp70-dependent. The capacity of the nucleolus to store misfolded proteins was limited, and prolonged stress led to a transition of the nucleolar matrix from liquid-like to solid, with loss of reversibility and dysfunction in quality control. Thus, we suggest that the nucleolus has chaperone-like properties and can promote nuclear protein maintenance under stress.

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