Kunio Nakatsukasa

Hydroxyurea (HU) has been extensively used in laboratory settings to induce S-phase arrest and checkpoint activation. Furthermore, it has a history of clinical use as a cost-effective chemotherapeutic agent. Nevertheless, there is still uncertainty regarding its precise pharmacology, side effects, and toxicity, particularly in terms of its impact on organelle homeostasis. Here, we demonstrate that in budding yeast, HU specifically inhibits the endoplasmic reticulum–associated degradation (ERAD) of luminal misfolded proteins (ERAD-L pathway), an effect that is independent of S-phase arrest. In contrast, HU did not affect the degradation of misfolded ER membrane proteins or the degradation of cytosolic proteins. The selective inhibition of ERAD-L by HU is likely attributable to the formation of disulfide bonds in cysteine residues in luminal substrates, which must be reduced before their retrotranslocation to the cytosol. We further demonstrate that HU plays a role in alleviating reductive stress phenotypes observed in cells lacking Ero1, which is essential for oxidative protein folding in the ER. We propose that HU functions as a modulator of thiol–disulfide homeostasis in the ER lumen.

Degradation of defective or superfluous mitochondria via mitophagy, a specialized form of selective autophagy, is important for maintaining mitochondrial quality and quantity. In yeast, the pro-mitophagic factor Atg32 is transcriptionally upregulated upon oxidative stress and anchored to the mitochondrial surface, where it acts as a molecular signal to initiate efficient degradation of mitochondria. However, how the protein levels of Atg32 are regulated post-translationally remains enigmatic. Here we show that the endoplasmic reticulum (ER) serves as a hub to govern Atg32 protein turnover. We found that the ER-associated degradation (ERAD) E3 ligase Doa10 interacts with Atg32, leading to its degradation by the proteasome. Furthermore, we show that Atg32 is destined for the ER in a manner dependent on the GET (guided entry of tail-anchored proteins) pathway, which mediates the delivery of tail-anchored (TA) proteins to the ER where Atg32 is potentially recognized by Doa10. Notably, Doa10 deficiency increased Atg32 levels and enhanced mitophagy under respiratory conditions, thus determining that ERAD serves as a brake on mitophagy.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of non-native Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-nucleoporin component of the central core of the NPC) was stable, C-terminally green fluorescent protein (GFP)-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of non-tagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of non-native nucleoporins generated by genetic mutation or when NPC biogenesis is impaired.

Most nucleus‐encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post‐translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCF^Ucc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCF^Ucc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCF^Ucc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

Misfolded luminal and membrane proteins in the endoplasmic reticulum (ER) are recognized and retrotranslocated to the cytosol for proteasomal degradation, a process referred to as ER-associated degradation (ERAD). In Saccharomyces cerevisiae , ERAD substrates with luminal lesions are targeted for proteasomal degradation by the Hrd1 ubiquitin ligase complex (ERAD-L pathway). Membrane proteins containing lesions within their membrane-spanning regions are also targeted for degradation by the Hrd1 complex (ERAD-M pathway), while those containing lesions within their cytosolic regions are targeted for degradation mainly by the Doa10 ubiquitin ligase complex (ERAD-C pathway). Here, we demonstrate that hydroxyurea (HU), which is widely used to arrest cells in S-phase and is also used to manage several diseases including sickle cell anemia and chronic myeloproliferative disorders, inhibited ERAD-L, but not ERAD-M or -C. HU-mediated inhibition of ERAD-L occurred independently of S-phase arrest. In HU-treated cells, the integrity of the Hrd1 ubiquitin ligase complex remained intact and substrate recognition was unaffected. Moreover, induction of the unfolded protein response was undetectable in cells in which ERAD-L was inhibited by HU. These results suggest an unexpected action of HU, which modulates protein quality control in the secretory pathway, and also suggest the existence of an additional regulatory step in ERAD.

In yeast, mitochondria are passed on to daughter cells via the actin cable, motor protein Myo2, and adaptor protein Mmr1. They are released from the actin-myosin machinery after reaching the daughter cells. We report that Mmr1 is rapidly degraded by the ubiquitin-proteasome system in Saccharomyces cerevisiae. Redundant ubiquitin ligases Dma1 and Dma2 are responsible for Mmr1 ubiquitination. Dma1/2-mediated Mmr1 ubiquitination requires phosphorylation, most likely at S414 residue by Ste20 and Cla4. These kinases are mostly localized to the growing bud and nearly absent from mother cells, ensuring phosphorylation and ubiquitination of Mmr1 after the mitochondria enter the growing bud. In dma1Δ dma2Δ cells, transported mitochondria are first stacked at the bud-tip and then pulled back to the bud-neck. Stacked mitochondria in dma1Δ dma2Δ cells exhibit abnormal morphology, elevated respiratory activity, and increased level of reactive oxygen species, along with hypersensitivity to oxidative stresses. Collectively, spatiotemporally-regulated Mmr1 turnover guarantees mitochondrial homeostasis.