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

  • Yuzy Matsuo
    Footnotes
    Affiliations
    From the Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan and
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  • Hayafumi Kishimoto
    Affiliations
    From the Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan and
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  • Katsuhiro Tanae
    Affiliations
    From the Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan and
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  • Kenji Kitamura
    Affiliations
    the Center for Gene Science, Hiroshima University, Kagamiyama 1-4-2, Higashi-Hiroshima 739-8527, Japan
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  • Satoshi Katayama
    Affiliations
    From the Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan and
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  • Makoto Kawamukai
    Correspondence
    To whom correspondence should be addressed: Dept. of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan. Tel.: 81-852-32-6583; Fax: 81-852-32-6092;
    Affiliations
    From the Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan and
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  • Author Footnotes
    * This work was supported by Japan Society for the Promotion of Science research fellowships for young scientists.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S4.
    1 Supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science.
Open AccessPublished:February 15, 2011DOI:https://doi.org/10.1074/jbc.M110.169953
      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.

      Introduction

      In eukaryotic cells, the ubiquitin-proteasome system (UPS)
      The abbreviations used are: UPS, ubiquitin-proteasome system; PQC, protein quality control; ER, endoplasmic reticulum; ts, temperature-sensitive; Ubi, ubiquitin; CHX, cycloheximide.
      has a pivotal role in multiple cellular events, including the cell cycle, signal transduction, and receptor-mediated endocytosis (
      • Hershko A.
      ,
      • Hochstrasser M.
      ,
      • Ciechanover A.
      ). 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) (
      • Goldberg A.L.
      ). 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 (
      • Shastry B.S.
      ). The removal of these harmful proteins and the maintenance of homeostasis are accomplished through the selective degradation of aberrant or misfolded proteins by the UPS. The involvement of the UPS in PQC in the cytoplasm and in the endoplasmic reticulum (ER) have been well studied, and several E3s for PQC have been identified, including Hrd1 and Doa10, which are involved in ER-associated degradation (
      • Hampton R.Y.
      ), as well as CHIP (
      • Murata S.
      • Chiba T.
      • Tanaka K.
      ) and Ubr1 (
      • Heck J.W.
      • Cheung S.K.
      • Hampton R.Y.
      ), both of which are associated with cytoplasmic PQC.
      In contrast, the study of nuclear PQC has been very limited to date. The lack of activity of the autophagy-lysosome system in the nucleus (
      • Iwata A.
      • Christianson J.C.
      • Bucci M.
      • Ellerby L.M.
      • Nukina N.
      • Forno L.S.
      • Kopito R.R.
      ) suggests that nuclear PQC depends entirely on the UPS. Supporting this hypothesis, studies demonstrated that the San1 E3 ligase, which shows specificity for nuclear aberrant proteins, plays a pivotal role in nuclear PQC in the budding yeast Saccharomyces cerevisiae (
      • Dasgupta A.
      • Ramsey K.L.
      • Smith J.S.
      • Auble D.T.
      ,
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ). Furthermore, recent studies in mammalian cells and primary neurons suggest that the UHRF-2 E3 ligase is an essential molecule for nuclear polyglutamine degradation as a component of the nuclear PQC machinery (
      • Iwata A.
      • Nagashima Y.
      • Matsumoto L.
      • Suzuki T.
      • Yamanaka T.
      • Date H.
      • Deoka K.
      • Nukina N.
      • Tsuji S.
      ). The high cellular toxicity of aberrant nuclear protein aggregates and their likely association with the neurodegenerative pathology of Huntington disease underscores the importance of investigating the role of nuclear PQC (
      • Shastry B.S.
      ).
      In the present study, the existence of a nuclear PQC system that functions through the UPS is demonstrated in the fission yeast Schizosaccharomyces pombe. A mutant form of Asf1 (Asf1-30) was used as a substrate for the study of the nuclear PQC system. Asf1 (anti-silencing function 1) was originally identified as a protein whose overexpression inhibits the silencing of chromatin in S. cerevisiae (
      • Le S.
      • Davis C.
      • Konopka J.B.
      • Sternglanz R.
      ) and subsequently as a complex with histones H3/H4 that could facilitate the replication-coupled assembly of nucleosomes by CAF1 in vitro (
      • Tyler J.K.
      • Adams C.R.
      • Chen S.R.
      • Kobayashi R.
      • Kamakaka R.T.
      • Kadonaga J.T.
      ). Asf1 is now recognized as a histone chaperone in a wide variety of eukaryotes, including humans (
      • Munakata T.
      • Adachi N.
      • Yokoyama N.
      • Kuzuhara T.
      • Horikoshi M.
      ). The results of the present study show that Ubc4 (E2) and San1 (E3) are the enzymes catalyzing the degradation of the Asf1-30 mutant protein. This is the first report identifying the molecules responsible for nuclear PQC in S. pombe.

      DISCUSSION

      The results of the present study show that the selective degradation of nuclear aberrant proteins mediates nuclear PQC in the fission yeast S. pombe. Although nuclear PQC has been reported in budding yeast and mammals (
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ,
      • Iwata A.
      • Nagashima Y.
      • Matsumoto L.
      • Suzuki T.
      • Yamanaka T.
      • Date H.
      • Deoka K.
      • Nukina N.
      • Tsuji S.
      ,
      • von Mikecz A.
      ), the molecular mechanisms of nuclear PQC in other organisms, including S. pombe, have not been elucidated. The present work demonstrates the instability of the mutant nuclear protein Asf1-30 at higher temperatures and its selective degradation by the ubiquitin-proteasome system. Although the present work only addresses the instability of Asf1-30, other nuclear mutant proteins (Mis12-537, Orc5-H37, Cnp1-1, Pim1-46, and Sad1-1) have also been found to be unstable at high temperatures (
      • Goshima G.
      • Iwasaki O.
      • Obuse C.
      • Yanagida M.
      ,
      • Kato H.
      • Matsunaga F.
      • Miyazaki S.
      • Yin L.
      • D'Urso G.
      • Tanaka K.
      • Murakami Y.
      ,
      • Chen E.S.
      • Saitoh S.
      • Yanagida M.
      • Takahashi K.
      ,
      • Matsumoto T.
      • Beach D.
      ,
      • Hagan I.
      • Yanagida M.
      ), suggesting that these mutant proteins could also be processed by the UPS. In fact, deletion of the san1 gene suppressed the temperature sensitivity of the mis12-537, cnp1-1, and pim1-46 mutants at semirestrictive temperature (Fig. 8, A–C). Furthermore, the instability of Cnp1-1 proteins was suppressed at the restrictive temperature in the san1 disruptant (Fig. 8D). These data suggested that San1 is involved in polyubiquitination and degradation of a variety of nuclear mutant proteins in S. pombe.
      The Ubc4 ubiquitin-conjugating enzyme (E2) was required for Asf1-30 degradation, and San1 was identified as the E3 ligase catalyzing the polyubiquitination of the Asf1-30 mutant protein. S. pombe Ubc4 is essential for growth and is responsible for the polyubiquitination of mitotic cyclin Cdc13 (
      • Seino H.
      • Kishi T.
      • Nishitani H.
      • Yamao F.
      ). Asf1-30 is therefore the second target of the E2 enzyme Ubc4 found to date. One unexpected finding was that different E2s mediate nuclear PQC in S. pombe and S. cerevisiae. In S. cerevisiae, the E2s Cdc34 and Ubc1 are associated with four distinct mutant nuclear proteins in the nuclear PQC (
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ). In the present work, the instability of Asf1-30 was not suppressed in S. pombe ubc15 (CDC34 orthologue) and ubc1 (UBC1 orthologue) mutants (Fig. 5A). Conversely, Ubc4 and Ubc5 (orthologues of Ubc4) are not required for degradation of mutant nuclear proteins in S. cerevisiae (
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ). Ubc4 and Ubc5 mediate the selective degradation of short lived and abnormal cytosolic proteins (
      • Seufert W.
      • Jentsch S.
      ). These results suggest that different E2 ubiquitin-conjugating enzymes are involved in the nuclear PQCs of S. cerevisiae and S. pombe.
      In contrast to the difference in nuclear PQC E2 enzymes between S. cerevisiae and S. pombe, the E3 enzyme San1 is active in both species. The san1 (sir antagonist) gene was originally identified as an extragenic suppressor of the sir4-9 mutant in S. cerevisiae (
      • Schnell R.
      • D'Ari L.
      • Foss M.
      • Goodman D.
      • Rine J.
      ), and it was later shown to possess ubiquitin ligase activity in vitro, and this activity was required for the in vivo function of San1 (
      • Dasgupta A.
      • Ramsey K.L.
      • Smith J.S.
      • Auble D.T.
      ). Recently, it was shown that San1 mediates the UPS involvement in the nuclear PQC of S. cerevisiae (
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ). In addition, ectopically expressed S. cerevisiae San1 enhanced the degradation of nuclear polyglutamine aggregates in cultured mammalian cells and thereby rescued polyglutamine-induced cytotoxicity (
      • Iwata A.
      • Nagashima Y.
      • Matsumoto L.
      • Suzuki T.
      • Yamanaka T.
      • Date H.
      • Deoka K.
      • Nukina N.
      • Tsuji S.
      ). Furthermore, recent reports show that the association between San1 and the E3 ligase Ubr1 is important for cytoplasmic PQC (
      • Heck J.W.
      • Cheung S.K.
      • Hampton R.Y.
      ). The present work used a different strategy to identify San1 as a ligase involved in the nuclear PQC of S. pombe by selecting six gene/proteins among 100 E3 candidates based on three criteria: nuclear localization, proteins containing an E3 domain, and the up-regulation of genes by heat shock. The S. pombe San1 E3 ligase was identified as the enzyme mediating the polyubiquitination of the Asf1-30 protein (Fig. 6, B and C). Although the role of San1 in targeting Asf1-30 was clear, the finding that the instability of Asf1-30 was not completely suppressed in a san1 mutant (Fig. 6A) compared with the ubc4ts mutant (Fig. 5A) suggests that other proteins might be involved in PQC. This would also explain the moderate suppression of temperature sensitivity of the asf1-30 allele by the san1 deletion (Fig. 7A). Intriguingly, the temperature sensitivity of the ubc4ts or mts2-1 mutants was enhanced by a concomitant asf1-30 mutation (Fig. S2). The accumulation of an Asf1-30 protein might have a deleterious effect in those ubc4ts and mts2-1 mutants characterized by an impaired degradation of aberrant and harmful proteins, emphasizing the importance of the nuclear PQC for the removal of damaging proteins and the maintenance of cellular homeostasis. Alternatively, stable Asf1-30 mutant protein in those ubc4ts and mts2-1 mutants might affect the process of other target proteins of UPS.
      Although the transcription of the san1 gene was up-regulated under heat shock stress (
      • Chen D.
      • Toone W.M.
      • Mata J.
      • Lyne R.
      • Burns G.
      • Kivinen K.
      • Brazma A.
      • Jones N.
      • Bähler J.
      ), the amount of San1 protein was not dramatically changed under these conditions (Fig. 9C). Because the San1 protein is stable in response to heat shock (Fig. 9D), a supraliminal amount of San1 might be sufficient to function in nuclear PQC. The finding that the san1 mutant did not show any obvious phenotypes under various different stress conditions tested (data not shown) suggests that other E3 ligases might function in nuclear PQC in S. pombe. These results are consistent with the finding that there is no obvious growth difference between wild type and the san1 mutant under various conditions in S. cerevisiae (
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ). Because San1 is the only E3 reported to function in nuclear PQC in two yeast types, it would be of great interest to find and characterize the other E3 ubiquitin ligases involved in nuclear PQC.
      The cellular compartment where polyubiquitinated Asf1-30 is degraded in S. pombe remains unknown. In S. pombe, the 26 S proteasome is enriched in the nucleus and nuclear periphery, both during interphase and the mitotic phase (
      • Wilkinson C.R.
      • Wallace M.
      • Morphew M.
      • Perry P.
      • Allshire R.
      • Javerzat J.P.
      • McIntosh J.R.
      • Gordon C.
      ,
      • Takeda K.
      • Yanagida M.
      ,
      • Tatebe H.
      • Yanagida M.
      ). The nuclear localization of Asf1-30 and San1 E3 ligase (FIGURE 4, FIGURE 9) suggests that polyubiquitinated Asf1-30 is degraded by the nuclear proteasome. However, the immunofluorescent signal of polyubiquitinated Asf1-30 was observed in the cytoplasm but not in the nucleus in the mts2-1 mutant at restrictive temperature (supplemental Fig. S3). These data suggested that polyubiquitinated Asf1-30 is not degraded in the nucleus but instead shunted out of the nucleus for degradation in the cytoplasm.
      As in S. pombe, the proteasome is enriched inside the nucleus throughout the cell cycle in S. cerevisiae (
      • Russell S.J.
      • Reed S.H.
      • Huang W.
      • Friedberg E.C.
      • Johnston S.A.
      ). In contrast to S. cerevisiae and S. pombe, the 26 S proteasome has been found in both the cytoplasm and nucleus of higher eukaryotic cells (
      • Wójcik C.
      • DeMartino G.N.
      ). Considering that the nucleus of yeasts is not broken down during cytokinesis, the nuclear localization of the 26 S proteasome is beneficial for the degradation of aberrant proteins generated in the cytoplasm and the nucleus in yeasts.
      The results presented in this study can be interpreted using the model shown in Fig. 10. At high temperature, the nuclear mutant protein Asf1-30 was selectively polyubiquitinated by the Uba1 E1 (ubiquitin-activating enzyme), Ubc4 E2 (a ubiquitin-conjugating enzyme), and San1 E3 (ubiquitin ligase) and degraded by the proteasome as a result of protein quality control in S. pombe. The present results suggest that the UPS functions in nuclear protein quality control in S. pombe and that nuclear PQC mediated by San1 and the UPS was evolutionarily conserved from S. cerevisiae to S. pombe. Although there are no apparent san1 orthologs in mammalian cells, analogous systems must exist in higher eukaryotes due to the importance of nuclear PQC in protecting non-dividing cells, such as neural and muscle cells, against the deleterious accumulation of nuclear protein aggregates. In fact, recent studies identified UHRF-2 as an essential E3 ubiquitin ligase involved in nuclear polyglutamine degradation as a component of the nuclear PQC in cultured cells and primary neurons (
      • Iwata A.
      • Nagashima Y.
      • Matsumoto L.
      • Suzuki T.
      • Yamanaka T.
      • Date H.
      • Deoka K.
      • Nukina N.
      • Tsuji S.
      ). This result indicates that nuclear PQC plays a key role in neuroprotection against the deleterious accumulation of nuclear protein aggregates in quiescent (G0) mammalian cells.
      Figure thumbnail gr10
      FIGURE 10E1-E2-E3 molecules that recognize a nuclear aberrant protein in S. pombe. At high temperatures, the nuclear localized mutant Asf1-30 protein was selectively polyubiquitinated by Uba1 (E1), Ubc4 (E2), and San1(E3) and degraded by the proteasome as a protein quality control system.
      Recent studies have suggested that S. pombe is an excellent model for the study of cellular quiescence, which can be achieved experimentally through nutritional limitation. Studies on the regulatory mechanism of G0 phase in S. pombe showed that the function of the proteasome is required for the maintenance of G0 quiescence (
      • Takeda K.
      • Yanagida M.
      ). The proteasome signal persisted in the nuclear periphery in G0 phase in S. pombe, but it diminished with the increase in cytoplasmic localization (
      • Takeda K.
      • Yanagida M.
      ,
      • Laporte D.
      • Salin B.
      • Daignan-Fornier B.
      • Sagot I.
      ). Furthermore, dysfunction of the proteasome in G0 phase caused the appearance of aberrant nuclear structures (
      • Takeda K.
      • Yoshida T.
      • Kikuchi S.
      • Nagao K.
      • Kokubu A.
      • Pluskal T.
      • Villar-Briones A.
      • Nakamura T.
      • Yanagida M.
      ). These features of S. pombe could help elucidate the physiological significance of the nuclear PQC in G0 phase, which would lead to a better understanding of the nuclear PQC in G0 phase in higher eukaryotes. In conclusion, nuclear PQC systems are active in budding yeast, fission yeast, and mammals, and they involve different E2s and E3s in each species.

      Acknowledgments

      We thank Drs. J. Bahler, T. Toda, H. Seino, F. Yamao, H. Yamano, and M. Yanagida and National Bio Resource Project (NBRP)/Yeast Genetic Resource Center (YGRC) for providing the materials and strains used in this study. We also thank Drs. H. Katoh, K. Nishimura, T. Kainou, T. Nakagawa, Y. Nagano, T. Anai, K. Watanabe, and our laboratory members for helpful discussions and experimental support.

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