Draft genome sequence of thermotolerant Saccharomyces cerevisiae AH465, isolated from Mt. Tatsuda, Kumamoto, Japan.

Thermotolerance is a required characteristic for biofuel production by yeast. Here, we report the draft genome sequence of thermotolerant Saccharomyces cerevisiae AH465, which was originally isolated by the authors. A hybrid assembly approach using MinION Mk1b and MiSeq sequencers was conducted. The assembled sequence comprises 13.4 Mb in 26 contigs.

Spathaspora quercus f.a., sp. nov., a novel yeast isolated from rotting wood and a lichen sample in Kyushu region, Japan.

Four novel d-xylose assimilation yeast strains were isolated from rotting wood and a lichen sample collected in the Kyushu region of Japan. Species identifications were performed by analysing the internal transcribed spacer 5.8S region sequences and the D1/D2 variable domain of the large subunit rRNA gene. Phylogenetic analysis suggested that these isolates are closely related to Spathaspora species isolated in China, such as S. jiuxiensis and S. parajiuxiensis. These isolates also showed sequence similarity to deposited sequences labelled as Schwanniomyces. They did not produce asci and ascospores under any of the test conditions. Based on phylogenetic analysis and phenotypic differences, Spathaspora quercus f.a., sp. nov. is proposed to accommodate these isolates. The holotype of Spathaspora quercus f.a., sp. nov. is NBRC 116146T (CBS18366). This species is able to ferment d-xylose, and a d-xylose fermentation test revealed that this species produces a considerable amount of xylitol.

Effect of yeast chromosome II aneuploidy on malate production in sake brewing.

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S. cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance.

Amine-Regulated pri-SMTP Oxidation in SMTP Biosynthesis in Stachybotrys: Possible Implication in Nitrogen Acquisition.

SMTP (the name SMTP is derived from Stachybotrys microspora triprenyl phenol) is a family of triprenyl phenol secondary metabolites from a black mold, Stachybotrys microspora. Some SMTP congeners exhibit anti-inflammatory and profibrinolytic activities that, in combination, contribute to the treatment of ischemic stroke. The final step in the SMTP biosynthesis is a non-enzymatic amine conjugation with an o-phthalaldehyde moiety of the precursor pre-SMTP, which can form adducts with proteins and nucleic acids. Thus, pre-SMTP formation should be a precisely regulated, rate-limiting step in the SMTP biosynthesis. To address the mechanism backing this regulation, we purified a metabolite that rapidly disappeared following amine feeding, identifying a novel compound, pri-SMTP. Furthermore, an enzyme, designated as pri-SMTP oxidase, responsible for pri-SMTP conversion to pre-SMTP, was purified. The formation of pri-SMTP, which is regulated by nitrogen and carbon nutrients, occurred in particular septate mycelia. Although pri-SMTP oxidase was expressed constitutively, the consumption of pri-SMTP was accelerated only when a primary amine was fed. Thus, SMTP biosynthesis is regulated by at least three mechanisms: (i) pri-SMTP formation affected by nutrients, (ii) the compartmentalization of pri-SMTP formation/storage, and (iii) amine-regulated pri-SMTP oxidation. Amine-regulated SMTP formation (i.e., amine-capturing with pre-SMTP) may play a role in the nitrogen acquisition/assimilation strategy in S. microspora, since pri-SMTP synthesis occurs on non-preferred nitrogen.

An overview of applications of CRISPR-Cas technologies in biomedical engineering.

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.

Systematic approach for assessing whether undeletable chromosomal regions in Saccharomyces cerevisiae are required for cell viability.

Previously, we identified 49 undeletable chromosomal regions harboring only non-essential genes in the genome of Saccharomyces cerevisiae. We proposed that there might be unknown synthetic lethal combinations of genes present in such undeletable regions of the genome. In this study, we chose four of the smallest undeletable chromosomal regions among the 49 and performed extensive further analyses to narrow down the gene-pairs responsible for lethality by replacing sub-regions in various combinations with a DNA module comprising the CgLEU2 marker. Although the methodology was different from previous study, interestingly the results revealed that not only the sub-regions but also the entire region was replaceable. To solve the apparent discrepancy between previous and present results, we further conducted additional analysis including investigation of suppressor mutation and mini-chromosome loss assay through the construction of mini-chromosome harboring two particular chromosomal regions with marked with URA3 marker by employing 5-FOA system. Based upon careful observation on the phenotype of colony formation on 5-FOA medium by spot test, we came to an important conclusion that particular chromosomal regions harboring only non-essential genes can be categorized into three classes, i.e., essential, non-essential and intrinsically essential. Intrinsically essential region is defined as appearance of papillae after mini-chromosome loss which implicates that the region is essential but compensatable against cell lethality. Our present study indicates that prudent and multiple approaches as performed in this study are needed to judge whether a particular chromosomal region of the S. cerevisiae genome is essential, non-essential or intrinsically essential but compensatable.

CRISPR-PCDup: a novel approach for simultaneous segmental chromosomal duplication in Saccharomyces cerevisiae.

In our previous study, a novel genome engineering technology, PCR-mediated chromosome duplication (PCDup), was developed in Saccharomyces cerevisiae that enabled the duplication of any desired chromosomal region, resulting in a segmental aneuploid. From one round of transformation, PCDup can duplicate a single chromosomal region efficiently. However, simultaneous duplication of multiple chromosomal regions is not possible using PCDup technology, which is a serious drawback. Sequential duplication is possible, but this approach requires significantly more time and effort. Because PCDup depends upon homologous recombination, we reasoned that it might be possible to simultaneously create duplications of multiple chromosomal regions if we could increase the frequency of these events. Double-strand breaks have been shown to increase the frequency of homologous recombination around the break point. Thus, we aimed to integrate the genome editing tool CRISPR/Cas9 system, which induces double-strand breaks, with our conventional PCDup. The new method, which we named CRISPR-PCDup increased the efficiency of a single duplication by up to 30 fold. CRISPR-PCDup enabled the simultaneous duplication of long chromosomal segments (160 kb and 200 kb regions). Moreover, we were also able to increase the length of the duplicated chromosome by up to at least 400 kb, whereas conventional PCDup can duplicate up to a maximum of 300 kb. Given the enhanced efficiency of chromosomal segmental duplication and the saving in both labor and time, we propose that CRISPR-PCDup will be an invaluable technology for generating novel yeast strains with desirable traits for specific industrial applications and for investigating genome function in segmental aneuploid.

CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae.

Genome manipulation, especially the deletion or replacement of chromosomal regions, is a salient tool for the analysis of genome function. Because of low homologous recombination activity, however, current methods are limited to manipulating only one chromosomal region in a single transformation, making the simultaneous deletion or replacement of multiple chromosomal regions difficult, laborious, and time-consuming. Here, we have developed two highly efficient and versatile genome engineering technologies, named clustered regularly interspaced short palindromic repeats (CRISPR)-PCR-mediated chromosomal deletion (PCD) (CRISPR-PCD) and PCR-mediated chromosomal replacement (CRISPR-PCRep), that integrate the CRISPR-associated protein 9 (Cas9) genome editing system (CRISPR/Cas9) into, respectively, the PCD method for chromosomal deletion and our newly developed PCRep method for chromosomal replacement. Integration of CRISPR induces double strand breaks to activate homologous recombination, and thus enhances the efficiency of deletion by PCD and replacement by PCRep, enabling multiple chromosomal regions to be manipulated simultaneously for the first time. Our data show that CRISPR-PCD can delete two internal or terminal chromosomal regions, while CRISPR-PCRep can replace triple chromosomal regions simultaneously in a single transformation. Colony PCR analysis of structural alterations showed that triple replacement of four different sets of chromosomal regions was successful in 83%-100% of transformants analyzed. These novel genome engineering technologies, which greatly reduce time and labor for genome manipulation, will provide powerful tools to facilitate the simultaneous multiple deletion and replacement of chromosomal regions, enabling the rapid analysis of genome function and breeding of useful industrial yeast strains.

gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.

Genetic analysis of suppressor mutants of a pho84 disruptant in the search for genes involved in intracellular inorganic phosphate sensing in Saccharomyces cerevisiae.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator.

As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400-700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae.

Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four independent sites in the genome can be split by one transformation. Total time and labor for obtaining a multiple split yeast strain is drastically reduced when compared with conventional PCS technology.

CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae.

PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function.

Improved stress resistance and ethanol production by segmental haploidization of the diploid genome in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains.

Genome-wide construction of a series of designed segmental aneuploids in Saccharomyces cerevisiae.

Segmental aneuploidy can play an important role in environmental adaptation. However, study of segmental aneuploids is severely hampered by the difficulty of creating them in a designed fashion. Here, we describe a PCR-mediated chromosome duplication (PCDup) technology that enables the generation of segmental aneuploidy at any desired chromosomal region in Saccharomyces cerevisiae. We constructed multiple strains harboring 100 kb to 200 kb segmental duplications covering the whole of the S. cerevisiae genome. Interestingly, some segmental aneuploidies confer stress tolerance, such as to high temperature, ethanol and strong acids, while others induce cell lethality and stress sensitivity, presumably as result of the simultaneous increases in dosages of multiple genes. We suggest that our PCDup technology will accelerate studies into the phenotypic changes resulting from alteration of gene dosage balance of multiple genes and will provide new insights into the adaptive molecular mechanisms in the genome in segmental aneuploidy-derived human diseases.

Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii.

We identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering in Saccharomyces cerevisiae.

Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

Type 2C protein phosphatase Ptc6 participates in activation of the Slt2-mediated cell wall integrity pathway in Saccharomyces cerevisiae.

The phosphorylation status of cellular proteins results from an equilibrium between the activities of protein kinases and protein phosphatases (PPases). Reversible protein phosphorylation is an important aspect of signal transduction that regulate many biological processes in eukaryotic cells. The Saccharomyces cerevisiae genome encodes 40 PPases, including seven members of the protein phosphatase 2C subfamily (PTC1 to PTC7). In contrast to other PPases, the cellular roles of PTCs have not been investigated in detail. Here, we sought to determine the cellular role of PTC6 in S. cerevisiae with disruption of PTC genes. We found that cells with Δptc6 disruption were tolerant to the cell wall-damaging agents Congo red (CR) and calcofluor white (CFW); however, cells with simultaneous disruption of PTC1 and PTC6 were very sensitive to these agents. Thus, simultaneous disruption of PTC1 and PTC6 gave a synergistic response to cell wall damaging agents. The level of phosphorylated Slt2 increased significantly after CR treatment in Δptc1 cells and more so in Δptc1Δptc6 cells; therefore, deletion of PTC6 enhanced Slt2 phosphorylation in the Δptc1 disruptant. The level of transcription of KDX1 upon exposure to CR increased to a greater extent in the Δptc1Δptc6 double disruptant than the Δptc1 single disruptant. The Δptc1Δptc6 double disruptant cells showed normal vacuole formation under standard growth conditions, but fragmented vacuoles were present in the presence of CR or CFW. Our analyses indicate that S. cerevisiae PTC6 participates in the negative regulation of Slt2 phosphorylation and vacuole morphogenesis under cell wall stress conditions.

The protein phosphatase Siw14 controls caffeine-induced nuclear localization and phosphorylation of Gln3 via the type 2A protein phosphatases Pph21 and Pph22 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14.