Hsp70–Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation

Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70–Bcl-2–associated athanogene 3 (Hsp70–Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70–Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70–Bag3–LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligo-mers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70–Bag3 complex therefore functions as an important signaling node that senses proteo-toxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation

An intrinsically disordered yeast prion arrests the cell cycle by sequestering a spindle pole body component

Intrinsically disordered proteins play causative roles in many human diseases. Their overexpression is toxic in many organisms, but the causes of toxicity are opaque. In this paper, we exploit yeast technologies to determine the root of toxicity for one such protein, the yeast prion Rnq1. This protein is profoundly toxic when overexpressed but only in cells carrying the endogenous Rnq1 protein in its [RNQ[superscript +]] prion (amyloid) conformation. Surprisingly, toxicity was not caused by general proteotoxic stress. Rather, it involved a highly specific mitotic arrest mediated by the Mad2 cell cycle checkpoint. Monopolar spindles accumulated as a result of defective duplication of the yeast centrosome (spindle pole body [SPB]). This arose from selective Rnq1-mediated sequestration of the core SPB component Spc42 in the insoluble protein deposit (IPOD). Rnq1 does not normally participate in spindle pole dynamics, but it does assemble at the IPOD when aggregated. Our work illustrates how the promiscuous interactions of an intrinsically disordered protein can produce highly specific cellular toxicities through illicit, yet highly specific, interactions with the proteome

A first order phase transition mechanism underlies protein aggregation in mammalian cells

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).National Institutes of Health (U.S.) (Grant DP2CA195769

Reinvestigation of aminoacyl-TRNA synthetase core complex by affinity purification-mass spectrometry reveals TARSL2 as a potential member of the complex

10.1371/journal.pone.0081734PLoS ONE812-POLN

A first order phase transition mechanism underlies protein aggregation in mammalian cells

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates

Prion Formation and Polyglutamine Aggregation Are Controlled by Two Classes of Genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.National Institutes of Health (U.S.) (grant GM56350)National Institutes of Health (U.S.) (NSRA F32 postdoctoral fellowship GM072340)National Institutes of Health (U.S.) (grant GM25874)Howard Hughes Medical Institut

Huntingtin toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1–151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1–85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity

Yeast Prions: Protein Aggregation Is Not Enough

Although many proteins -- both damaged and normal -- have a tendency to aggregate, only some are capable of dividing and propagating. What does it take to turn a protein aggregate into an infectious prion