An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control

In this paper, we show that the essential Hsp90 co-chaperone Sgt1 is a member of a general protein quality control network that links folding and degradation through its participation in the degradation of misfolded proteins both in the cytosol and the endoplasmic reticulum (ER). Sgt1-dependent protein degradation acts in a parallel pathway to the ubiquitin ligase (E3) and ubiquitin chain elongase (E4), Hul5, and overproduction of Hul5 partly suppresses defects in cells with reduced Sgt1 activity. Upon proteostatic stress, Sgt1 accumu- lates transiently, in an Hsp90- and proteasome-dependent manner, with quality control sites (Q-bodies) of both yeast and human cells that co-localize with Vps13, a protein that creates organelle contact sites. Misfolding disease proteins, such as synphilin-1 involved in Parkinson's disease, are also sequestered to these compartments and require Sgt1 for their clearance

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regard less of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7,gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, high-lighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the proteindisaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was nothindered when pro3-1 colocalized with a slowly resolved mis-folded protein. Finally, we observed using super-resolutionlight microscopy as well as immunogold labeling EM in which both showed an even distribution of the different mis-folded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compart-mentalization, allows for differential clearance of these mis-folding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

Genetically controlled mtDNA deletions prevent ROS damage by arresting oxidative phosphorylation

Deletion of mitochondrial DNA in eukaryotes is currently attributed to rare accidental events associated with mitochondrial replication or repair of double-strand breaks. We report the discovery that yeast cells arrest harmful intramitochondrial superoxide production by shutting down respiration through genetically controlled deletion of mitochondrial oxidative phosphorylation genes. We show that this process critically involves the antioxidant enzyme superoxide dismutase 2 and two-way mitochondrial-nuclear communication through Rtg2 and Rtg3. While mitochondrial DNA homeostasis is rapidly restored after cessation of a short-term superoxide stress, long-term stress causes maladaptive persistence of the deletion process, leading to complete annihilation of the cellular pool of intact mitochondrial genomes and irrevocable loss of respiratory ability. This shows that oxidative stress-induced mitochondrial impairment may be under strict regulatory control. If the results extend to human cells, the results may prove to be of etiological as well as therapeutic importance with regard to age-related mitochondrial impairment and disease

On the role of actin in yeast protein quality control

Every cell is equipped with a protein quality control system to ensure the proper function of proteins. This is essential for both cell maintenance and the generation of new and healthy cells. In this thesis, the budding yeast Saccharomyces cerevisiae is used as a model to study both spatial quality control and the management of the protein involved in Huntington’s disease. The role of the actin cytoskeleton in both these processes has been the special focus of the thesis. Earlier studies established a role for the histone deacetylase Sir2 and the actin cytoskeleton in the asymmetrical inheritance of damaged proteins by the mother cell, as cells either lacking SIR2 or subjected to a transient collapse of the actin cytoskeleton, fail in this segregation process. In this thesis the protein disaggregase Hsp104, the polarisome complex, and the molecular chaperone CCT were identified as additional factors having important functions in the asymmetric segregation of damaged proteins. CCT is an essential, cytosolic folding machine, vital for the production of native actin. The actin folding capacity of CCT appears to be regulated by Sir2. Without this regulation the cell suffers from a reduction in native actin molecules, which could affect the integrity of actin cytoskeletal structures. The polarisome complex ensures actin polymerization at the bud tip and the establishment of a retrograde actin cable flow from the bud to the mother. Our data show that the presence of a functional actin cytoskeleton allows for Hsp104, associated with protein aggregates, to use the actin cytoskeleton as a scaffold and prevent the inheritance of damaged and aggregated proteins by the daughter. The retention of damaged protein within the mother cell is important for the rejuvenation of the daughter cell, as a daughter being born with increased damage suffer from a reduced life span

Defects in β-cell Ca2+ dynamics in age-induced diabetes

Little is known about the molecular mechanisms underlying age-dependent deterioration in β-cell function. We now demonstrate that age-dependent impairment in insulin release, and thereby glucose homeostasis, is associated with subtle changes in Ca2+ dynamics in mouse β-cells. We show that these changes are likely to be accounted for by impaired mitochondrial function and to involve phospholipase C/inositol 1,4,5-trisphosphate–mediated Ca2+ mobilization from intracellular stores as well as decreased β-cell Ca2+ influx over the plasma membrane. We use three mouse models, namely, a premature aging phenotype, a mature aging phenotype, and an aging-resistant phenotype. Premature aging is studied in a genetically modified mouse model with an age-dependent accumulation of mitochondrial DNA mutations. Mature aging is studied in the C57BL/6 mouse, whereas the 129 mouse represents a model that is more resistant to age-induced deterioration. Our data suggest that aging is associated with a progressive decline in β-cell mitochondrial function that negatively impacts on the fine tuning of Ca2+ dynamics. This is conceptually important since it emphasizes that even relatively modest changes in β-cell signal transduction over time lead to compromised insulin release and a diabetic phenotype.Accepted versio

Snd3 controls nucleus-vacuole junctions in response to glucose signaling

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites

Nuclear Hsp104 safeguards the dormant translation machinery during quiescence

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished

Nuclear envelope budding is a response to cellular stress

Nuclear envelope budding (NEB) is a recently discovered alternative pathway for nucleocytoplasmic communication distinct from the movement of material through the nuclear pore complex. Through quantitative electron microscopy and tomography, we demonstrate how NEB is evolutionarily conserved from early protists to human cells. In the yeast Saccharomyces cerevisiae, NEB events occur with higher frequency during heat shock, upon exposure to arsenite or hydrogen peroxide, and when the proteasome is inhibited. Yeast cells treated with azetidine-2-carboxylic acid, a proline analog that induces protein misfolding, display the most dramatic increase in NEB, suggesting a causal link to protein quality control. This link was further supported by both localization of ubiquitin and Hsp104 to protein aggregates and NEB events, and the evolution of these structures during heat shock. We hypothesize that NEB is part of normal cellular physiology in a vast range of species and that in S. cerevisiae NEB comprises a stress response aiding the transport of protein aggregates across the nuclear envelope