p62-containing,proteolytically active nuclear condensates,increase the efficiency of the ubiquitin–proteasome system

Degradation of a protein by the ubiquitin–proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them—a ubiquitin ligase (E3)—belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid–liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.

An important, yet unsolved question is how cells recruit the appropriate reactants involved in biochemical reactions, to a defined location at a specific time to increase the reaction efficiency and processivity. This is particularly relevant for sequential transformations along cascades that are catalyzed by different enzymes and complexes, such as degradation of a protein by the ubiquitin–proteasome system (UPS). Separation of the interior milieu of membrane-bound organelles (e.g., nucleus, Golgi apparatus, endoplasmic reticulum, mitochondria, and lysosomes) from the surrounding environment enables the cells to control the spatial distribution of specific reactions and their components. However, this compartmentalization makes it difficult to allow exchange of components necessary for providing high specificity, for example. In addition, cells contain supramolecular assemblies that are not bound by surrounding membranes and can be found both in the cytoplasm (such as the centriole, P bodies, and stress granules) and the nucleus (such as nucleoli, promyelocytic leukemia [PML] bodies, Cajal bodies, and nuclear speckles) (1, 2).Emerging evidence shows that many membraneless biomolecular condensates are formed by liquid–liquid phase separation (LLPS) (3). These condensates provide a different environment from the surrounding cytoplasm or nucleoplasm and probably have an important role in the concentration of biomolecules, thus allowing efficient multistep processes to occur. In addition, it allows for rapid exchange of specific components—such as ubiquitin ligases—to occur and thus allowing accommodation to changing pathophysiological conditions and the requirement for high specificity of different processes. Recently, it was shown that p62, also known as Sequestosome-1 (SQSTM-1), forms spherical liquid-like condensates (bodies) both in the cytosol and in a cell-free system (4). Formation of these condensates is mediated by the ability of the protein to oligomerize into helical filaments through its N-terminal PB1 (Phox and Bem1) domain, and to bind ubiquitin (Ub) chains attached to target substrates via its C-terminal UBA (ubiquitin-associated) domain (5). The efficiency of p62 condensate formation was shown to be strongly dependent on the length of the Ub chains attached to the substrates (6).Cytosolic p62 condensates play an important role in maintaining protein homeostasis and quality control by their ability to sequester ubiquitinated proteins and shuttle them for degradation by the autophagy–lysosome system. This is mediated via direct interaction of the p62’s LIR motif with the autophagosome-incorporated LC3 (7). In addition to its role in autophagy, p62 was shown to deliver cytosolic ubiquitinated substrates to the proteasome, by direct interaction of its PB1 domain with the 19S Rpn1 and Rpn10 proteasomal subunits (8).p62 is widely distributed in the cell, and its nucleocytoplasmic shuttling is mediated by two nuclear localization signals (NLS1 and NLS2) and one nuclear export signal (NES) (9). In addition to its cytosolic functions, p62 forms nuclear bodies that exist both as separate and as hybrid structures by interacting with PML or Cajal bodies (10). Since autophagy occurs exclusively in the cytosol, the role and biological significance of nuclear p62 have remained elusive.In the present study we show that p62 is involved in the formation of nuclear LLP-separated foci, which recruit components of the UPS and protein substrates, resulting in their efficient degradation under both basal and stressed conditions.