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...

The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe.

The msa2/nrd1 gene encodes an RNA-binding protein that negatively regulates sexual differentiation of fission yeast Schizosaccharomyces pombe by repressing the Ste11-regulated genes. However, it is not known how Msa2 regulates sexual differentiation, and to characterize its role, we altered the msa2 gene by inducing point mutations and tested the resulting mutants for their ability to inhibit sexual differentiation and their suppressive effect on a temperature sensitive pat1 mutant. Several amino acids were found to be important, including three phenylalanine residues (F153, F245 and F453) in the three consensus RNA recognition motifs (RRMs) and a threonine residue (T126) that normally functions as a phosphorylation site. Results indicated that Msa2 was negatively regulated by phosphorylation that arose from Spk1-mediated pheromone signaling. Msa2 also regulated the Ste11 protein level coordinating with Cpc2, a ribosomal-associated protein. In addition, Msa2 was detected in stress granules that co-localized with Pabp in the cytosol under conditions of glucose starvation. Msa2 may regulate the translation of Ste11, be a component of stress granules that form in response to glucose starvation, and regulate the sexual differentiation of S. pombe.

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a b s t r a c tAn H3/H4 histone chaperone, Asf1, plays an essential role in maintaining genomic stability in many species, including fission yeast. Here, we showed that overexpression of a CENP-A chaperone Sim3 suppressed the temperature sensitive phenotype of asf1-33 and asf1-30 mutants and the defect in chromatin structure, and prevented the accumulation of DNA damage in asf1-33 mutants at high temperatures. Furthermore, asf1-33 and Dsim3 were synthetic lethal. Consistent with this, shutdown of sim3 expression in asf1-33 Dsim3 double mutants that contained extragenic sim3 resulted in growth retardation. In addition, the Dsim3 mutant displayed sensitivity to thiabendazol and hydroxyurea, which suggests that Sim3 plays a general role in maintaining chromatin structure. Our results suggest a possibility that Sim3 functions as a histone chaperone.