Protein homeostasis, or proteostasis, is crucial for the functioning of a cell, as proteins that are mislocalized, present in excessive amounts, or aberrant due to misfolding or other type of damage can be harmful. Proteostasis includes attaining the correct protein structure, localization, and the formation of higher order complexes, and well as the appropriate protein concentrations. Consequences of proteostasis imbalance are evident in a range of neurodegenerative diseases characterized by protein misfolding and aggregation, such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis. To protect the cell from the accumulation of aberrant proteins, a network of protein quality control (PQC) pathways identifies the substrates and direct them towards refolding or elimination via regulated protein degradation. The main pathway for degradation of misfolded proteins is the ubiquitin-proteasome system. PQC pathways have been first described in the cytoplasm and the endoplasmic reticulum, however, accumulating evidence indicates that the nucleus is an important PQC compartment for ubiquitination and proteasomal degradation of not only nuclear, but also cytoplasmic proteins. In this review, we summarize the nuclear ubiquitin-proteasome pathways involved in proteostasis maintenance in yeast, focusing on inner nuclear membrane-associated degradation (INMAD) and San1-mediated protein quality control.

An intricate machinery protects cells from the accumulation of misfolded, non-functional proteins and protein aggregates. Protein quality control pathways have been best described in the cytoplasm and the endoplasmic reticulum, however, recent findings indicate that the nucleus is also an important compartment for protein quality control. Several nuclear ubiquitinylation pathways target soluble and membrane proteins in the nucleus and mediate their degradation through nuclear proteasomes. In addition, emerging data suggest that nuclear envelope components are also degraded by autophagy, although the mechanisms by which cytoplasmic autophagy machineries get access to nuclear targets remain unclear. In this minireview we summarize the nuclear ubiquitin-proteasome pathways in yeast, focusing on pathways involved in the protein degradation at the inner nuclear membrane. In addition, we discuss potential mechanisms how nuclear targets at the nuclear envelope may be delivered to the cytoplasmic autophagy pathways in yeast and mammals.

Many cells spend a major part of their life in quiescence, a reversible state characterized by a distinct cellular organization and metabolism. In glucose-depleted quiescent yeast cells, there is a metabolic shift from glycolysis to mitochondrial respiration, and a large fraction of proteasomes are reorganized into cytoplasmic granules containing disassembled particles. Given these changes, the operation of protein quality control (PQC) in quiescent cells, in particular the reliance on degradation-mediated PQC and the specific pathways involved, remains unclear. By examining model misfolded proteins expressed in glucose-depleted quiescent yeast cells, we found that misfolded proteins are targeted for selective degradation requiring functional 26S proteasomes. This indicates that a significant pool of proteasomes remains active in degrading quality control substrates. Misfolded proteins were degraded in a manner dependent on the E3 ubiquitin ligases Ubr1 and San1, with Ubr1 playing a dominant role. In contrast to exponentially growing cells, the efficient clearance of certain misfolded proteins additionally required intact nucleus-vacuole junctions (NVJ) and Cue5-independent selective autophagy. Our findings suggest that proteasome activity, autophagy, and NVJ-dependent degradation operate in parallel. Together, the data demonstrate that quiescent cells maintain active PQC that relies primarily on selective protein degradation. The necessity of multiple degradation pathways for the removal of misfolded proteins during quiescence underscores the importance of misfolded protein clearance in this cellular state.

Background: The primary histological characteristic of Alzheimer's disease is the presence of neurofibrillary tangles, which are large aggregates of tau protein. Aging is the primary risk factor for the development of Alzheimer's disease, however, the underlying causes of tau protein aggregation and toxicity are unclear.Aims: Here we investigated tau aggregation and toxicity under the conditions of compromised protein homeostasis.Methods: We used heterologous expression of human tau protein in the unicellular eukaryote yeast Saccharomyces cerevisiae with evolutionarily conserved protein quality control pathways and examined tau‐dependent toxicity and aggregation using growth assays, fluorescence microscopy, and a split luciferase‐based reporter NanoBiT.Results: Tau protein expressed in yeast under mild proteotoxic stress, or in mutants with impaired pathways for proteotoxic stress response, did not lead to synthetic toxicity or the formation of obvious aggregates. Chronologically old cells also did not develop observable tau aggregates. Our examination of tau oligomerization in living cells using NanoBiT reporter suggests that tau does not form significant levels of oligomers under basal conditions or under mild proteotoxic stress.Conclusion: Together our data suggest that human tau protein does not represent a major burden to the protein quality control system in yeast cells.

Recent data suggest that early stages of Alzheimer’s disease (AD) are characterized by an abnormally high phosphorylation of microtubule-associated protein tau and truncation of its C-terminus. Tau hyperphosphorylation may result from the downregulation of phosphatases, especially protein phosphatase 2A. In an attempt to model and analyze these molecular events we treated SH-SY5Y cells with okadaic acid (OA), an inhibitor of protein phosphatases. In addition to the low molecular weight tau, such treatment lead to the appearance of heat-stable protein species with apparent high molecular weight around 100 kDa, which were immunoreactive to tau antibodies against phosphorylated Ser202 and phosphorylated Ser396. Based on the observation that high molecular weight tau-immunoreactive proteins (HMW-TIP) correspond to the predicted size of two monomers of tau, one possibility is that HMW-TIP represent tau oligomers. The absence of HMW-TIP detection by anti-total tau antibodies used may be a consequence of epitope masking, or a combination of epitope masking and protein truncation. We noted the stability of HMW-TIP in the presence of strong denaturing agents, such as urea and guanidine, as well as upon partial dephosphorylation by the alkaline phosphatase. Moreover, as HMW-TIP did not dissociate the presence of β-mercaptoethanol, it was also independent from disulfide bonds. Taken together, these data show that OA treatment of SH-SY5Y cells induces the appearance of HMW-TIP, which may represent tau oligomer or tau-crossreactive phospho-proteins. Our findings have implications for further studies of tau under the conditions of protein phosphatase downregulation.

Quiescence is a conserved, reversible state of proliferative arrest, characterized by changes in cell physiology and metabolism. Many cells spend a considerable part of their lifetime in quiescence, including adult stem cells or microorganisms facing unfavorable environmental conditions. Cells can remain quiescent for long periods of time while retaining their viability and reproductive capacity, indicating a need to maintain protein homeostasis. Given the changes in intracellular organization, it has been unclear how protein quality control (PQC) functions in quiescent cells. In our recent study, we examined model misfolded proteins expressed in glucose-depleted quiescent yeast cells and found that quiescent cells maintain an active PQC that relies primarily on selective protein degradation, requiring the ubiquitin-proteasome system, intact nucleus-vacuole junctions and autophagy. Our results highlight the relevance of mitigating misfolded proteins in quiescence.