Search

The facilitated aggregation of misfolded proteins is a proteostasis strategy important for cell function and viability, but the molecular mechanisms are poorly understood. Grousl et al. reveal how the intrinsically disordered domains of the small heat shock protein Hsp42 promote and control the aggregation of misfolded proteins during stress conditions in yeast., Chaperones with aggregase activity promote and organize the aggregation of misfolded proteins and their deposition at specific intracellular sites. This activity represents a novel cytoprotective strategy of protein quality control systems; however, little is known about its mechanism. In yeast, the small heat shock protein Hsp42 orchestrates the stress-induced sequestration of misfolded proteins into cytosolic aggregates (CytoQ). In this study, we show that Hsp42 harbors a prion-like domain (PrLD) and a canonical intrinsically disordered domain (IDD) that act coordinately to promote and control protein aggregation. Hsp42 PrLD is essential for CytoQ formation and is bifunctional, mediating self-association as well as binding to misfolded proteins. Hsp42 IDD confines chaperone and aggregase activity and affects CytoQ numbers and stability in vivo. Hsp42 PrLD and IDD are both crucial for cellular fitness during heat stress, demonstrating the need for sequestering misfolded proteins in a regulated manner.

Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension—the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation. DOI: http://dx.doi.org/10.7554/eLife.11794.001, eLife digest Cells use molecular machines called ribosomes to build proteins by connecting amino acids – the building blocks of proteins – together in a particular sequence. The chain of amino acids gradually lengthens as the protein forms, yet remains attached to the ribosome until the protein is complete. While this process is underway, cells can check that a newly forming chain is not abnormal or damaged. If it is, a cell then essentially ‘decides’ on whether to correct or eliminate it. Such protein quality control processes are important for ensuring the health and fitness of cells and organisms. Recently, a new protein quality control mechanism was discovered that senses when a ribosome becomes jammed as it produces a new protein. This mechanism recycles the ribosome so it can make more new proteins. It also disposes of the stalled protein using a cell complex, called the ribosome-associated quality control complex, which is found in all eukaryotic organisms including yeast and humans. This protein complex consists of three subunits; one of which, called Rcq2, tags ribosome-stalled proteins with a “tail” that contains the amino acids alanine and threonine. However, the purpose of this tag was not clear. Yonashiro, Tahara et al. now show that the tagging of ribosome-stalled proteins by Rqc2 in yeast cells induces the tagged proteins to clump together. This clumping probably prevents these proteins from inadvertently interfering with other molecules or processes within the cell. The formation of these clumps also correlates with the activation of a stress response in the cell, indicating that these clumps create a signal that prompts the cell to protect itself in response to the accumulation of more abnormal proteins. Mutations in one subunit of the ribosome-associated quality control complex in mice cause a condition that resembles a neurological disease in humans, called amyotrophic lateral sclerosis or ALS for short. A future challenge is therefore to understand how much Rqc2-mediated tagging and clumping of ribosome-stalled protein has a role in this and other neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.11794.002

[ISP+] is a prion form of the global transcriptional regulator Sfp1 in Saccharomyces cerevisiae that manifests phenotypically as an antisuppressor of specific sup35 nonsense suppressor mutations. Although SUP35 is a Sfp1 target, the mechanism of antisuppression is unclear. Here we show that the level of SUP35 transcription in [ISP+] cells containing the sup35 mutation is increased relative to [isp-] cells and cells with a SFP1 deletion. As a result, [ISP+] cells have increased amounts of Sup35 encoded by the mutant allele. Indeed, additional experiments showed that increased amounts of mutant Sup35 may cause antisuppression. Remarkably, [ISP+] effects are not equivalent to those produced by SFP1 deletion, so [ISP+] represents an obvious example of a functionally active prion form of a protein. This feature distinguishes [ISP+] from other yeast prions, where prion switch often has the same effect as inactivation of a prion host gene. We suggest that enhancement of SUP35 expression in [ISP+] cells is caused by specific interaction of Sfp1 in its prion form with some negative SUP35 regulator. We also demonstrate that the advantage of [ISP+] strains over [isp-] strains described in our earlier work is specific for certain genetic background and growth conditions.

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone-assisted nuclear import via nuclear pores. The compartment-specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter-compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.