Ellagitannins and Oligomeric Proanthocyanidins of Three Polygonaceous Plants.

The aim of this study was to characterize hydrolyzable tannins in Polygonaceous plants, as only a few plants have previously been reported to contain ellagitannins. From Persicaria chinensis, a new hydrolyzable tannin called persicarianin was isolated and characterized to be 3-O-galloyl-4,6-(S)-dehydrohexahydroxydiphenoyl-d-glucose. Interestingly, acid hydrolysis of this compound afforded ellagic acid, despite the absence of a hexahydroxydiphenoyl group. From the rhizome of Polygonum runcinatum var. sinense, a large amount of granatin A, along with minor ellagitannins, helioscpoinin A, davicratinic acids B and C, and a new ellagitannin called polygonanin A, were isolated. Based on 2D nuclear magnetic resonance (NMR) spectroscopic examination, the structure of polygonanin A was determined to be 1,6-(S)-hexahydroxydiphenoyl-2,4-hydroxychebuloyl-ß-d-glucopyranose. These are the second and third hydrolyzable tannins isolated from Polygonaceous plants. In addition, oligomeric proanthocyanidins of Persicaria capitatum and P. chinensis were characterized by thiol degradation. These results suggested that some Polygonaceous plants are the source of hydrolyzable tannins not only proanthocyanidins.

Highly Oxidized Ellagitannins of Carpinus japonica and Their Oxidation-Reduction Disproportionation.

In the research on ellagitannin metabolism, two unique dehydroellagitannins, carpinins E (1) and F (2), bearing dehydrohexahydroxydiphenoyl (DHHDP) and hydrated biscyclohexenetrione dicarboxyl ester (HBCHT) groups, were isolated from young leaves of Carpinus japonica. Upon heating in H2O or treatment with pH 6 buffer at room temperature, 1 and 2 afforded the reduction product 3, isocarpinin A, with an (R)-hexahydroxydiphenoyl (HHDP) group, suggesting the occurrence of redox disproportionation of the (S)-DHHDP group. This was supported by the increase in production of 3 in the pH 6 buffer solution by coexistence of epigallocatechin-3-O-gallate (15), accompanied by oxidation of 15. In contrast, treatment of 1 and 2 with ascorbic acid yielded 4, carpinin A, with an (S)-HHDP group. Upon heating with ascorbic acid, the HBCHT group was also reduced to an (S)-HHDP group, and 2 was converted to 2,3;4,6-bis(S)-HHDP glucose. In leaves of C. japonica, the tannins 1 and 2 are dominant in young spring leaves, but compounds 3 and 4 become the major components of tannins in mature leaves. These results suggest that, in ellagitannin biosynthesis, oxidative coupling of the two galloyl groups first generates a DHHDP group, and subsequent reduction of DHHDP esters produces HHDP esters.

Fermentative Production of Cysteine by Pantoea ananatis.

Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe.IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production.

Improved fermentative L-cysteine overproduction by enhancing a newly identified thiosulfate assimilation pathway in Escherichia coli.

Sulfate (SO42-) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (S2O32-), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO32-) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S2-) â†’ L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.

Reduced capacity of alternative sigmas to melt promoters ensures stringent promoter recognition.

In bacteria, multiple sigmas direct RNA polymerase to distinct sets of promoters. Housekeeping sigmas direct transcription from thousands of promoters, whereas most alternative sigmas are more selective, recognizing more highly conserved promoter motifs. For sigma(32) and sigma(28), two Escherichia coli Group 3 sigmas, altering a few residues in Region 2.3, the portion of sigma implicated in promoter melting, to those universally conserved in housekeeping sigmas relaxed their stringent promoter requirements and significantly enhanced melting of suboptimal promoters. All Group 3 sigmas and the more divergent Group 4 sigmas have nonconserved amino acids at these positions and rarely transcribe >100 promoters. We suggest that the balance of "melting" and "recognition" functions of sigmas is critical to setting the stringency of promoter recognition. Divergent sigmas may generally use a nonoptimal Region 2.3 to increase promoter stringency, enabling them to mount a focused response to altered conditions.

Bacterial Cysteine-Inducible Cysteine Resistance Systems.

UNLABELLED: Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA(formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded byccdR(formerly PAJ_0332 orybaO), located just upstream of ccdA The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR(formerly PAJ_3027), located just upstream of cefA To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra- or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis IMPORTANCE: Because of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and efflux, the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for industrial purposes. We propose that this novel mechanism for sensing and regulating cysteine levels is a safety valve enabling adaptation to sudden changes in intra- or extracellular cysteine levels in bacteria. Our findings provide important insights into optimizing the production of cysteine and related biomaterials by P. ananatis and also a deep understanding of sulfur/cysteine metabolism and regulation in this plant pathogen and related bacteria.

Induction of the Escherichia coli yijE gene expression by cystine.

Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess cystine. Transcriptome analysis showed 11 up-regulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one up-regulated membrane gene, yijE, and two down-regulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli.

Roxbin B is cuspinin: structural revision and total synthesis.

Prompted by the outcome that the synthesized roxbin B was not identical to the natural roxbin B, the structural determination process and spectral data were re-examined, with the finding that roxbin B was very likely to be 1-O-galloyl-2,3-(R);4,6-(S)-bis-O-hexahydroxydiphenoyl-ß-d-glucose (cuspinin). Because the (R)-axial chirality is rare in natural products when the hexahydroxydiphenoyl group bridges the 2- and 3-oxygens, the proposed structure of cuspinin was confirmed by the total synthesis, leading to the conclusion that roxbin B is the same as cuspinin.

Yellow pigments produced by oxidative oligomerization of dihydrochalcone glucoside and the reaction mechanism.

From the dried leaves of Lithocarpus polystachyus, yellow pigments, lithocarputins B (11) and C (12), were isolated with a colorless dihydrochalcone dimer, lithocarputin A (10). The pigments 11 and 12 are dimeric dihydrochalcone glycosides with bicyclo[3.2.1]octane structures. Each pigment is a diastereomeric mixture with enantiomeric aglycones that could not be separated. The production mechanisms of the pigments were proposed based on the in vitro enzymatic preparation from trilobatin (1), the major dihydrochalcone glucoside of L. polystachyus. The majority of the pigments in the dried leaves were the oligomers of the dihydrochalcone glycosides generated by a mechanism similar to dimerization. The pigments are probably artifacts produced in the drying process. This is the first report disclosing a detailed chemical mechanism for pigment formation from dihydrochalcone.

Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli.

BACKGROUND: Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. RESULTS: Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. CONCLUSIONS: In this work, we showed that Grx1 and NrdH reduce SSC to L-cysteine, and the generated sulfite is then utilized as the sulfur source to produce additional L-cysteine molecule through the sulfate pathway in E. coli. We also found that co-overexpression of NrdH, CysI, and CysK increases L-cysteine production. Our results propose that the enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from SSC is a novel method for improvement of L-cysteine production.

Microbial Production Potential of Pantoea ananatis: From Amino Acids to Secondary Metabolites.

Pantoea ananatis, a gram-negative bacterium belonging to the Erwiniaceae family, is a well-known phytopathogen isolated from many ecological niches and plant hosts. However, this bacterium also provides us with various beneficial characteristics, such as the growth promotion of their host plants and increased crop yield. Some isolated non-pathogenic strains are promising for the microbial production of useful substances. P. ananatis AJ13355 was isolated as an acidophilic bacterium and was used as an excellent host to produce L-glutamic acid under acidic conditions. The genome sequence of P. ananatis AJ13355 was determined, and specific genome-engineering technologies were developed. As a result, P. ananatis was successfully used to construct a bacterial strain that produces cysteine, a sulfur-containing amino acid that has been difficult to produce through fermentation because of complex regulation. Furthermore, by heterologous expression including plant-derived genes, construction of a strain that produces isoprenoids such as isoprene and linalool as secondary metabolites was achieved. P. ananatis is shown to be a useful host for the production of secondary metabolites, as well as amino acids, and is expected to be used as a platform for microbial production of bioactive substances, aromatic substances, and other high-value-added substances of plant origin in the future.

Fermentative production of sulfur-containing amino acid with engineering putative l-cystathionine and l-cysteine uptake systems in Escherichia coli.

Here, proteins involved in sulfur-containing amino acid uptake in Escherichia coli strains were investigated with the aim of applying the findings in fermentative amino acid production. A search of genes in an l-methionine auxotrophic strain library suggested YecSC as the putative transporter of l-cystathionine. l-Methionine production increased by 15% after amplification of yecSC in producer strains. A candidate protein responsible for l-cysteine uptake was also found by experimentation with multicopy suppressor E. coli strains that recovered from growth defects caused by l-cysteine auxotrophy. Based on the results of an uptake assay, growth using l-cysteine as a sole sulfur source, and sensitivity to l-cysteine toxicity, we proposed that YeaN is an l-cysteine transporter. l-Cysteine production increased by 50% as a result of disrupting yeaN in producer strain. The study of amino acid transporters is valuable to industrialized amino acid production and also sheds light on the role of these transporters in sulfur assimilation.

Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake.

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica.

The sigma(E)-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. sigma(E) is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the sigma(E) regulon is driven from a suite of approximately 60 sigma(E)-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the sigma(E) promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (-35 to -65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the alpha-subunit of RNA polymerase; (ii) ppGpp generally activates sigma(E) promoters, and (iii) sigma(E) promoters are responsive to changing sigma(E) holoenzyme levels under physiological conditions, reinforcing the idea that the sigma(E) regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

Conserved and variable functions of the sigmaE stress response in related genomes.

Bacteria often cope with environmental stress by inducing alternative sigma (sigma) factors, which direct RNA polymerase to specific promoters, thereby inducing a set of genes called a regulon to combat the stress. To understand the conserved and organism-specific functions of each sigma, it is necessary to be able to predict their promoters, so that their regulons can be followed across species. However, the variability of promoter sequences and motif spacing makes their prediction difficult. We developed and validated an accurate promoter prediction model for Escherichia coli sigmaE, which enabled us to predict a total of 89 unique sigmaE-controlled transcription units in E. coli K-12 and eight related genomes. SigmaE controls the envelope stress response in E. coli K-12. The portion of the regulon conserved across genomes is functionally coherent, ensuring the synthesis, assembly, and homeostasis of lipopolysaccharide and outer membrane porins, the key constituents of the outer membrane of Gram-negative bacteria. The larger variable portion is predicted to perform pathogenesis-associated functions, suggesting that sigmaE provides organism-specific functions necessary for optimal host interaction. The success of our promoter prediction model for sigmaE suggests that it will be applicable for the prediction of promoter elements for many alternative sigma factors.

Preparation, characterization, and antioxidative effects of oligomeric proanthocyanidin-L-cysteine complexes.

Controlled acid-catalyzed degradation of proanthocyanidin polymers in grape seeds together with L-cysteine led to oligomeric proanthocyanidin-L-cysteine complexes along with monomeric flavan-3-ol derivatives being isolated, and their structures were confirmed on the basis of spectroscopic data and by chemical means. In addition, comparative studies on the antioxidative and survival effects of oligomeric proanthocyanidin-L-cysteine complexes and proanthocyanidin polymers were performed. The oligomeric proanthocyanidin-L-cysteine complexes showed higher bioavailability and antioxidant capacity and enhanced survival time in the animal test groups. In addition, it is suggested that the oligomeric complexes may help to prevent oxidative stress and may reduce free radical production.

Protective effect of persimmon peel polyphenol against high glucose-induced oxidative stress in LLC-PK(1) cells.

The effect of persimmon peel polyphenol (PPP) on high glucose-induced oxidative stress was investigated using LLC-PK(1) cells, which is susceptible to oxidative stress. High-concentration glucose (30 mM) treatment induced LLC-PK(1) cell death, but high molecular-PPP (HMPPP) and low molecular-PPP (LMPPP), at concentrations of 5 or 10 microg/ml, significantly inhibited the high glucose-induced cytotoxicity. Furthermore, treatment with HMPPP or LMPPP dose-dependently reduced the intracellular reactive oxygen species level increased by 30 mM glucose. In addition, nitric oxide, superoxide and peroxynitrite levels were increased by 30 mM glucose treatment, but they were concentration-dependently inhibited by HMPPP or LMPPP treatment. High glucose levels induced the overexpressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins, but HMPPP or LMPPP treatment reduced the overexpressions of these proteins. HMPPP or LMPPP also inhibited the nuclear translocation of nuclear factor-kappa B (NF-kappaB) induced by 30 mM glucose in LLC-PK(1) cells. In particular, LMPPP exhibited stronger inhibitory activities on high glucose induced oxidative stress than HMPPP. These findings indicate the potential benefits of persimmon peel as a valuable source of antioxidants in the diabetic condition which will reduce the oxidative stress induced by hyperglycemia.

Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli.

Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

ydjN encodes an S-sulfocysteine transporter required by Escherichia coli for growth on S-sulfocysteine as a sulfur source.

Sulfur is an essential element for growth and many physiological functions. As sulfur sources for Escherichia coli and related bacteria, specific transporters import various sulfur-containing compounds from the environment. In this study, we identified and characterized an alternative function of the cystine transporter YdjN in E. coli as a transporter of S-sulfocysteine, a sulfur-containing intermediate in the assimilatory cysteine biosynthesis that is used as a sulfur source for the growth of E. coli We also demonstrated that the transport of S-sulfocysteine via YdjN depends on the transcriptional regulator CysB, a master regulator that controls most of the genes involved in sulfur assimilation and cysteine metabolism. We found that the use of S-sulfocysteine as a sulfur source depends on glutathione because mutations in glutathione biosynthetic genes abolish growth when S-sulfocysteine is used as a sole sulfur source, thereby supporting the previous findings that the conversion of S-sulfocysteine to cysteine is catalyzed by glutaredoxins. To the best of our knowledge, this is the first report of a functional S-sulfocysteine transporter across organisms, which strongly supports the hypothesis that S-sulfocysteine is not only a metabolic intermediate but also a physiologically significant substance in specific natural environments.

Isolation of Saccharomyces cerevisiae RNase T1 hypersensitive (rns) mutants and genetic analysis of the RNS1/DSL1 gene.

Overexpression of the rntA cDNA encoding RNase T1 derived from A. oryzae causes severe growth inhibition in S. cerevisiae. We previously reported that most S. cerevisiae mutant strains defective in translocation into the ER, ER-Golgi transport and vacuole formation exhibited hypersensitivity to expression of RNase T1. Screening for S. cerevisiae mutants that showed RNase T1 hypersensitivity resulted in the isolation of 38 (rns) mutant strains. Some of these mutants showed a variety of phenotypes including temperature-sensitive growth, hypersensitivity to G418, defect in invertase glycosylation and fragmented vacuoles. We identified the genes mutated in three of the rns mutants, rns1, rns2, and rns3, as DSL1, UMP1, and SEC17, respectively. Fluorescence microscopic observation showed that GFP or myc-tagged Rns1p was localized at the nuclear region in the cell. Two-hybrid screening revealed the interaction of Rns1p with a transcription factor Cin5p and a functionally unknown Ylr440cp. It was observed that HA-tagged Ylr440cp was localized to the ER and nuclear envelope.