Stress- and ubiquitylation-dependent phase separation of the proteasome. Nature The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation. 10.1038/s41586-020-1982-9

Phase Separation, Transition, and Autophagic Degradation of Proteins in Development and Pathogenesis. Wang Zheng,Zhang Hong Trends in cell biology Phase separation and transition control the assembly and material states (liquid, gel like, or solid) of protein condensates to ensure that distinct cellular functions occur in a spatiotemporally controlled manner. The assembly and biophysical properties of condensates are precisely regulated by chaperone proteins, post-translational modifications (PTMs), and numerous cellular factors. Phase separation also triages misfolded and unwanted proteins for autophagic degradation. The concerted actions of receptor proteins, scaffold proteins, and PTMs determine the size, assembly rate, and material properties of condensates for efficient removal. Altered phase separation and transition affect the degradation of protein condensates, resulting in their accumulation under certain developmental and pathological conditions. Elucidation of the role of phase separation and transition in the degradation of disease-related protein condensates will provide insights into the molecular mechanism underlying the pathogenesis of various diseases. 10.1016/j.tcb.2019.01.008

Liquid-liquid phase separation in autophagy. Noda Nobuo N,Wang Zheng,Zhang Hong The Journal of cell biology Liquid-liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into distinct condensates. Liquid-like condensates can transition into gel and solid states, which are essential for fulfilling their different functions. LLPS plays important roles in multiple steps of autophagy, mediating the assembly of autophagosome formation sites, acting as an unconventional modulator of TORC1-mediated autophagy regulation, and triaging protein cargos for degradation. Gel-like, but not solid, protein condensates can trigger formation of surrounding autophagosomal membranes. Stress and pathological conditions cause aberrant phase separation and transition of condensates, which can evade surveillance by the autophagy machinery. Understanding the mechanisms underlying phase separation and transition will provide potential therapeutic targets for protein aggregation diseases. 10.1083/jcb.202004062

Phase separation organizes the site of autophagosome formation. Fujioka Yuko,Alam Jahangir Md,Noshiro Daisuke,Mouri Kazunari,Ando Toshio,Okada Yasushi,May Alexander I,Knorr Roland L,Suzuki Kuninori,Ohsumi Yoshinori,Noda Nobuo N Nature Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. 10.1038/s41586-020-1977-6