Nuclear Protein Quality Is Regulated by the Ubiquitin-Proteasome System through the Activity of Ubc4 and San1 in Fission Yeast

Abstract

Eukaryotic cells monitor and maintain protein quality through a set of protein quality control (PQC) systems whose role is to minimize the harmful effects of the accumulation of aberrant proteins. Although these PQC systems have been extensively studied in the cytoplasm, nuclear PQC systems are not well understood. The present work shows the existence of a nuclear PQC system mediated by the ubiquitin-proteasome system in the fission yeast Schizosaccharomyces pombe. Asf1-30, a mutant form of the histone chaperone Asf1, was used as a model substrate for the study of the nuclear PQC. A temperature-sensitive Asf1-30 protein localized to the nucleus was selectively degraded by the ubiquitin-proteasome system. The Asf1-30 mutant protein was highly ubiquitinated at higher temperatures, and it remained stable in an mts2-1 mutant, which lacks proteasome activity. The E2 enzyme Ubc4 was identified among 11 candidate proteins as the ubiquitin-conjugating enzyme in this system, and San1 was selected among 100 candidates as the ubiquitin ligase (E3) targeting Asf1-30 for degradation. San1, but not other nuclear E3s, showed specificity for the mutant nuclear Asf1-30, but did not show activity against wild-type Asf1. These data clearly showed that the aberrant nuclear protein was degraded by a defined set of E1-E2-E3 enzymes through the ubiquitin-proteasome system. The data also show, for the first time, the presence of a nuclear PQC system in fission yeast.In eukaryotic cells, the ubiquitin-proteasome system (UPS) 3 has a pivotal role in multiple cellular events, including the cell cycle, signal transduction, and receptor-mediated endocytosis (1-3). This system is essential for the selective degradation of many cellular proteins. The substrate proteins destined for degradation are first recognized by the ubiquitination machinery, triggering the covalent attachment of a polyubiquitin chain to the target protein. Polyubiquitinated proteins are recognized and degraded by the 26 S proteasome. The formation of the polyubiquitin chain is accomplished by a series of enzymatic reactions catalyzed by three enzymes: an E1 (ubiquitin-activating enzyme), an E2 (ubiquitin-conjugating enzyme), and an E3 (ubiquitin ligase). The step catalyzed by the E3 is crucial in determining substrate selectivity and timing of degradation, which implies that the identification and understanding of E3s is important to elucidate the mechanisms of specific substrate selection.The UPS also contributes to cellular protein quality control (PQC) (4). Aberrant or misfolded proteins are produced in the cell by mutation or environmental stress. The intracellular accumulation of these proteins causes proteotoxic or harmful effects. In humans, for example, the accumulation of aberrant proteins is thought to be associated with diseases such as Alzheimer, Huntington, Parkinson, and Creutzfeldt-Jakob diseases (5). The removal of these harmful proteins and the maintenance of homeostasis are accomplished through the selective degradation of aberrant or misfolded proteins...

Human Cytomegalovirus UL76 Elicits Novel Aggresome Formation via Interaction with S5a of the Ubiquitin Proteasome System

“…S2 in the supplemental material) (58). One determinant of the initiation of protein misfolding appears to be the protein sequence, for which the transition from hidden to exposed hydrophobicity is critical and has to be recognized by specific E3 ligases of UPS, such as San1 in yeast (12)(13)(14)59). Although insoluble protein aggresomes are the hallmark of misfolded proteins, previous studies by Bennett and colleagues provide insight into the induction of dysfunctional protein folding.…”

“…In the cytoplasm, misfolded proteins are degraded by two different processes: the UPS and autophagy involved in lysosomal proteolysis. Because of the lack of autophagy, the clearing of misfolded proteins residing in the nucleus appears to be conducted entirely by the UPS (12)(13)(14). Failure to remove misfolded proteins elicits protein aggregation or deposition as insoluble aggresomes, which are associated with severe neurological diseases, including Creutzfeldt-Jakob disease.…”

How the Nucleus Copes with Proteotoxic Stress

“…The primary nuclear ubiquitin ligase is San1, which ubiquitylates misfolded polypeptide substrates for degradation in both budding and fission yeasts [73–75]. San1 localizes to the nucleus under stress and nonstress conditions [74, 75], and ubiquitylates a large variety of misfolded substrates, including destabilized endogenous mutant proteins, short synthetic polypeptides, exogenous model misfolded proteins, as well as misfolded ER substrates lacking their ER-targeting sequences [74, 76–81].…”

“…San1 localizes to the nucleus under stress and nonstress conditions [74, 75], and ubiquitylates a large variety of misfolded substrates, including destabilized endogenous mutant proteins, short synthetic polypeptides, exogenous model misfolded proteins, as well as misfolded ER substrates lacking their ER-targeting sequences [74, 76–81]. The subcellular localizations of these polypeptides are diverse, where they are found in the nucleus but, perhaps more surprisingly, also in the cytoplasm as soluble or ER membrane-associated states (more on this below).…”

Serum glycomarkers of endoplasmic reticulum and lysosomal-endosomal system stress in human healthy aging and diseases

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To verify the idea that extracellular free oligosaccharides might be able to reflect the functional status of the endoplasmic reticulum (ER) and lysosomal-endosomal system, HPLC-profiles of serum-derived free oligosaccharides (FOS) in human healthyT he multilayered protein quality assurance system encompasses almost all cellular organelles, a network of biochemical processes and a multiplex of intracellular and extracellular signaling cascades [1][2][3][4][5]. At the centre of this wide cellular landscape there are two main hubs -the endoplasmic reticulum (ER) and lysosomal-endosomal system.

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