Misfolded proteins and insoluble aggregates are continuously produced in the cell and can result in severe stress that threatens cellular fitness and viability if not managed effectively. Accordingly, organisms have evolved several protective protein quality control (PQC) machineries to address these threats. In eukaryotes, the ubiquitin-proteasome system (UPS) plays a vital role in the disposal of intracellular misfolded, damaged, or unneeded proteins. Although ubiquitin-mediated proteasomal degradation of many proteins plays a key role in the PQC system, cells must also dispose of the proteasomes themselves when their subunits are assembled improperly, or when they dysfunction under various conditions, e.g., as a result of genomic mutations, diverse stresses, or treatment with proteasome inhibitors. Here, we review recent studies that identified the regulatory pathways that mediate proteasomes sorting under various stress conditions, and the elimination of its dysfunctional subunits. Following inactivation of the 26S proteasome, UPS-mediated degradation of its own misassembled subunits is the favored disposal pathway. However, the cytosolic cell-compartment-specific aggregase, Hsp42 mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments, where they become extensively modified with ubiquitin, and are directed by ubiquitin receptors for autophagic clearance (proteaphagy). We also discuss the sorting mechanisms that the cell uses under nitrogen stress, and to distinguish between dysfunctional proteasome aggregates and proteasome storage granules (PSGs), reversible assemblies of membrane-free cytoplasmic condensates that form in yeast upon carbon starvation and help protect proteasomes from autophagic degradation. Regulated proteasome subunit homeostasis is thus controlled through cellular probing of the level of proteasome assembly, and the interplay between UPS-mediated degradation or sorting of misfolded proteins into distinct cellular compartments.
The vast majority of processes within the cell are carried out by proteins working in conjunction. The Yeast Two-Hybrid (Y2H) methodology allows the detection of physical interactions between any two interacting proteins. Here, we describe a novel systematic genetic methodology, "Reverse Yeast Two-Hybrid Array" (RYTHA), that allows the identification of proteins required for modulating the physical interaction between two given proteins. Our assay starts with a yeast strain in which the physical interaction of interest can be detected by growth on media lacking histidine, in the context of the Y2H methodology. By combining the synthetic genetic array technology, we can systematically screen mutant libraries of the yeast to identify-acting mutations that disrupt the physical interaction of interest. We apply this novel method in a screen for mutants that disrupt the interaction between the N-terminus of Elg1 and the Slx5 protein. Elg1 is part of an alternative replication factor C-like complex that unloads PCNA during DNA replication and repair. Slx5 forms, together with Slx8, a SUMO-targeted ubiquitin ligase (STUbL) believed to send proteins to degradation. Our results show that the interaction requires both the STUbL activity and the PCNA unloading by Elg1, and identify topoisomerase I DNA-protein cross-links as a major factor in separating the two activities. Thus, we demonstrate that RYTHA can be applied to gain insights about particular pathways in yeast, by uncovering the connection between the proteasomal ubiquitin-dependent degradation pathway, DNA replication, and repair machinery, which can be separated by the topoisomerase-mediated cross-links to DNA.
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