Identification ofsam4as arad24Allele inSchizosaccharomyces pombe

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...

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“…RNAs were prepared for RT-PCR as described previously (25). S. pombe cells were grown in YES medium at 36°C to a density of 1.0 ϫ 10 7 cells/ml.…”

Section: Rna Preparation and Rt-pcr-totalmentioning

confidence: 99%

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

Matsuo

Kishimoto

Tanae

et al. 2011

Journal of Biological Chemistry

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“…Screening of genes able to suppress the slow growth phenotype of an sam2 mutant Oowatari et al (2009) reported that 0.1 M CaCl 2 inhibited growth of an sam2 mutant. To identify sam2 + or multicopy suppressors of sam2 mutant, we screened for transformants of the sam2 mutant that were no longer sensitive to CaCl 2 .…”

Section: Resultsmentioning

confidence: 99%

“…Mutations in sam3 and sam 9 are dominant, while mutations in sam1 , sam2 , sam4 , sam5 , sam6 , sam7 , and sam8 are recessive (Katayama et al ., ). The sam4 mutation was reported to be allelic to rad24 (Oowatari et al ., ), while mutations in sam5 , sam6 , and sam7 mutants were found to be allelic to pka1 (Gupta et al ., , b). The sam2 , sam3 , sam4 , and sam9 mutants were shown to be sensitive for growth on medium containing 0.1 M Ca 2+ , suggesting involvement of Ca 2+ tolerance and sexual differentiation, but mutations caused by this phenotype have not been identified, except rad24 mutation in sam4 (Ozoe et al ., ; Oowatari et al ., ).…”

Section: Introductionmentioning

confidence: 99%

The zinc finger protein Gsf1 regulates Gsf2-dependent flocculation in fission yeast

et al. 2013

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Fission yeast flocculates nonsexually by induction of the flocculin encoded by gsf2(+) which is controlled by the positive regulator Mbx2. Here, we report a novel gene designated gsf1(+) found to be a negative regulator of nonsexual flocculation. We identified gsf1(+) as a multicopy suppressor of a sam2 mutation, which caused growth sensitivity to Ca(2+) and also found a nonsense mutation in gsf1(+) in a previously isolated gsf1 mutant. The gsf1(+) gene encodes a 547-aa protein containing a Zn(2)-Cys(6) binuclear cluster-type zinc finger motif. The Gsf1 protein localized in the nucleus, consistent with a role as a transcription factor. Deletion of gsf1(+) resulted in nonsexual flocculation inducible by CaCl2 , which was suppressed by the addition of EDTA or galactose. Both gsf2(+) and mbx2(+) were highly expressed in the gsf1 mutant. gsf1∆ gsf2∆ and gsf1∆ mbx2∆ double mutants did not flocculate, suggesting that gsf1(+) is an upstream regulator. In addition, the gsf1 mutant was sensitive to CaCl2 , KCl, HU, and TBZ, consistent with the possibility that gsf1(+) plays a role in functions unrelated to flocculation. Taken together, these results suggest that nonsexual flocculation in fission yeast is negatively controlled by Gsf1, which controls expression of mbx2(+) and gsf2(+) .

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“…Among nine sam mutants, this is the second example of identification of its mutant locus next to the identification of sam4 as a rad24 allele. 21) It has been found that the S. pombe Ápka1 cells are de-repressed for sexual differentiation even in the presence of rich nutrition, and that Ápka1 spores are apparently impaired in germination and often fail to resume vegetative growth. 10,39) These pka1 deletion mutant phenotypes were similarly observed in sam6, which was selected to undergo sexual differentiation under nutrient-rich conditions.…”

Section: Discussionmentioning

confidence: 99%

“…20) We identified sam4 as an allele of rad24, which encodes a 14-3-3 protein. 21) Besides this, we analyzed and characterized two suppressor genes, msa1 and msa2, 22,23) both of which encode RNA binding proteins that negatively regulate sexual differentiation. Through analysis of sam3 and sam9, we identified sla1 24,25) as an inducer of sexual differentiation, and zds1 as involving both sexual differentiation and CaCl 2 tolerance.…”

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confidence: 99%

Complex Formation, Phosphorylation, and Localization of Protein Kinase A ofSchizosaccharomyces pombeupon Glucose Starvation

Gupta

Paul

Oowatari

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.

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Identification ofsam4as arad24Allele inSchizosaccharomyces pombe

Cited by 18 publications

References 1 publication

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

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

The zinc finger protein Gsf1 regulates Gsf2-dependent flocculation in fission yeast

Complex Formation, Phosphorylation, and Localization of Protein Kinase A ofSchizosaccharomyces pombeupon Glucose Starvation

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