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• W2024253884 abstract "Congo red binds to the cell wall and inhibits the growth of yeast. In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1Δ mutant, we found that a previously uncharacterized gene, YBR005w, makes most of the Saccharomyces cerevisiae strains resistant to Congo red. This gene was named RCR1 (resistance to Congo red 1). An rcr1Δ null mutant showed an increased sensitivity to Congo red. RCR1 encodes a novel ER membrane protein with a single transmembrane domain. Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity. We examined the effect of RCR1 in various null mutants of genes related to the cell wall. The resistance of mutants to Congo red correlates with a reduction of chitin content. Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change. The binding of Calcofluor white to the cell wall significantly decreased in these cells. Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S. cerevisiae. Congo red binds to the cell wall and inhibits the growth of yeast. In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1Δ mutant, we found that a previously uncharacterized gene, YBR005w, makes most of the Saccharomyces cerevisiae strains resistant to Congo red. This gene was named RCR1 (resistance to Congo red 1). An rcr1Δ null mutant showed an increased sensitivity to Congo red. RCR1 encodes a novel ER membrane protein with a single transmembrane domain. Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity. We examined the effect of RCR1 in various null mutants of genes related to the cell wall. The resistance of mutants to Congo red correlates with a reduction of chitin content. Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change. The binding of Calcofluor white to the cell wall significantly decreased in these cells. Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S. cerevisiae. The fungal cell wall plays an important role in protecting the cell from various types of stress, including noxious chemicals and osmotic pressure. The cell wall of the budding yeast Saccharomyces cerevisiae is composed of β1,3- and β1,6-glucan, chitin, and mannoproteins (1Klis F.M. Mol P. Hellingwerf K. Brul S. FEMS Microbiol. Rev. 2002; 26: 239-256Crossref PubMed Google Scholar). About half of the cell wall is made up of β1,3-glucan that has linkage with other polymers. β1,6-Glucan mainly links mannoproteins to β1,3-glucan. Chitin, a linear polymer of β1,4-linked N-acetylglucosamine, constitutes only 2–3% of the cell wall but has a vital role in S. cerevisiae (2Shaw J.A. Mol P.C. Bowers B. Silverman S.J. Valdivieso M.H. Duran A. Cabib E. J. Cell Biol. 1991; 114: 111-123Crossref PubMed Scopus (361) Google Scholar). These components are under a dynamic and highly regulated control by stress or cell cycle and have a complementary role in which a decrease in one component is immediately compensated by an increase in others. In a defective mutant of fks1 that encodes a β1,3-glucan synthase catalytic subunit, the content of glucan greatly reduces, but the amount of chitin increases instead. Similar change in cell wall components was found in a gas1 mutant that releases soluble glucan in the medium and accumulates chitin and mannoproteins (3Ram A.F. Kapteyn J.C. Montijn R.C. Caro L.H. Douwes J.E. Baginsky W. Mazur P. van den Ende H. Klis F.M. J. Bacteriol. 1998; 180: 1418-1424Crossref PubMed Google Scholar, 4Popolo L. Gilardelli D. Bonfante P. Vai M. J. Bacteriol. 1997; 179: 463-469Crossref PubMed Google Scholar). The mutant yeast cell that has an altered cell wall composition by the compensating system shows a different response to the external stress from the wild-type cell. A significant case is the sensitivity to K1 killer toxin or Calcofluor white. These compounds have a specific target in cell wall components and therefore have been used in the study of the cell wall. K1 killer toxin binds to its receptor, including β1,6-glucan, and forms fatal ion channels in the plasma membrane (5Martinac B. Zhu H. Kubalski A. Zhou X.L. Culbertson M. Bussey H. Kung C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6228-6232Crossref PubMed Scopus (107) Google Scholar). A number of genes concerned in the synthesis of β1,6-glucan have been identified by studying the killer toxin-resistant (kre) mutants (6Boone C. Sommer S.S. Hensel A. Bussey H. J. Cell Biol. 1990; 110: 1833-1843Crossref PubMed Scopus (176) Google Scholar, 7Brown J.L. Kossaczka Z. Jiang B. Bussey H. Genetics. 1993; 133: 837-849Crossref PubMed Google Scholar). Calcofluor white preferentially binds to polysaccharides containing β1,4-linked d-glucopyranosyl units (8Wood P.J. Carbohydr. Res. 1980; 85: 271-287Crossref Scopus (289) Google Scholar). In yeast, it binds to chitin and alters the assembly of its microfibrils (9Elorza M.V. Rico H. Sentandreu R. J. Gen. Microbiol. 1983; 129: 1577-1582PubMed Google Scholar). Therefore, the sensitivity against this compound closely relates to the chitin content. Mutants with increased chitin, by compensating for the defect of other components such as fks1 or gas1, show higher sensitivity to Calcofluor white (10Ram A.F. Wolters A. Ten Hoopen R. Klis F.M. Yeast. 1994; 10: 1019-1030Crossref PubMed Scopus (272) Google Scholar, 11Ram A.F. Brekelmans S.S. Oehlen L.J. Klis F.M. FEBS Lett. 1995; 358: 165-170Crossref PubMed Scopus (122) Google Scholar). On the other hand, reduction in chitin content makes cells more resistant to Calcofluor white. The mutants chs3, which encodes a major chitin synthase, and chs4-chs7, which help in the proper localization or activation of Chs3, show higher resistance to Calcofluor white (12Trilla J.A. Duran A. Roncero C. J. Cell Biol. 1999; 145: 1153-1163Crossref PubMed Scopus (126) Google Scholar, 13Bulawa C.E. Mol. Cell. Biol. 1992; 12: 1764-1776Crossref PubMed Scopus (149) Google Scholar, 14Jablonowski D. Fichtner L. Martin V.J. Klassen R. Meinhardt F. Stark M.J. Schaffrath R. Yeast. 2001; 18: 1285-1299Crossref PubMed Scopus (51) Google Scholar). Similarly, overexpression of KNR4, which represses the chitin synthesis genes, makes the cell more resistant to Calcofluor white (15Martin H. Dagkessamanskaia A. Satchanska G. Dallies N. Francois J. Microbiology. 1999; 145: 249-258Crossref PubMed Scopus (33) Google Scholar). Although the stilbene-type dye Calcofluor white has been extensively used in many cell wall mutant studies, another cell wall-perturbing agent, benzidine-type dye Congo red, has not been widely used in identifying cell wall mutants. One of the reasons is that the effect of Congo red on the cell wall in S. cerevisiae is somehow ambiguous. Because Congo red stimulates chitin synthesis in S. cerevisiae like Calcofluor white (16Roncero C. Duran A. J. Bacteriol. 1985; 163: 1180-1185Crossref PubMed Google Scholar), both compounds are thought to have similar effects on fungal cell wall. But it has also been known that Congo red binds to β1,3-glucan (17Ogawa K. Hatano M. Carbohydr. Res. 1978; 67: 527-535Crossref Scopus (64) Google Scholar, 18Ogawa K. Tsurugi J. Watanabe T. Carbohydr. Res. 1973; 29: 397-403Crossref Scopus (90) Google Scholar) and interferes with the assembly of the β1,3-glucan filaments in S. cerevisiae (19Kopecka M. Gabriel M. Arch. Microbiol. 1992; 158: 115-126Crossref PubMed Scopus (127) Google Scholar). These effects would not be mutually exclusive, and the mechanism of growth inhibition by Congo red in S. cerevisiae remains somewhat unclear. Congo red as well as Calcofluor white binds to β1,4-linked d-glucopyranoside units, but the binding specificity to other polysaccharides, such as Curdlan, which mainly consists of β1,3-linked d-glucopyranoside units, was completely different from Calcofluor white (8Wood P.J. Carbohydr. Res. 1980; 85: 271-287Crossref Scopus (289) Google Scholar). Therefore, the effect may be different among biological species. So far, there is little information on the relationship between the sensitivity to Congo red and the genes that are concerned in cell wall synthesis of S. cerevisiae. In this work, we report characterization of a previously uncharacterized gene RCR1 (resistance to Congo red 1) and firm evidence indicating that the primary target of Congo red in S. cerevisiae is chitin rather than glucan. Strains and Media—Escherichia coli K12 strain DH5α (F-, φ80lacZΔM15, supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used in plasmid propagation, and Escherichia coli was grown in an LB (1% Bacto-Tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) medium with or without 100 μg/ml ampicillin. S. cerevisiae strains used in this study are listed in Table I. Yeast was grown in YPD medium (1% yeast extract (BD Biosciences), 2% peptone (BD Biosciences), 2% glucose) and SD medium (0.17% yeast nitrogen base without amino acids (BD Biosciences), 0.5% (NH4)2SO4, 2% glucose, and appropriate supplements) (20Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Solid media were made with 2% agar. Calcofluor white (Fluorescent Brightener 28, Sigma) and Congo red (Sigma) were added to the media at the concentrations indicated.Table IYeast strains used in this study Other deletion strains used in this study were obtained from EUROSCARF.StrainGenotypeSourceKA31MATa/α his3/his3 leu2/leu2 trp1/trp1 ura3/ura3Laboratory strainKA31-1AMATa his3 leu2 trp1 ura3Laboratory strainYKI59MATa his3 leu2 trp1 ura3 erd1Δ::LEU2This studyYKI75MATa his3 leu2 trp1 ura3 rcr1Δ::LEU2This studyBY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0EUROSCARFYKI89as BY4741, rcr1Δ::LEU2This studyYKI116-2as BY4741, CHS7::3HAThis studyYKI117-1as BY4741, rcr1Δ::LEU2 rcr2Δ::HIS3This studyYKI118-1as BY4741, rcr2Δ::HIS3This studyBY4741 chs7Δas BY4741, chs7Δ::kanMX4EUROSCARFBY4741 fks1Δas BY4741, fks1Δ::kanMX4EUROSCARFBY4741 pmt2Δas BY4741, pmt2Δ::kanMX4EUROSCARFBY4741 rom2Δas BY4741, rom2Δ::kanMX4EUROSCARFBY4741 rot2Δas BY4741, rot2Δ::kanMX4EUROSCARFBY4741 swi6Δas BY4741, swi6Δ::kanMX4EUROSCARFY1304MATa ura3-52 lys2-801 ade2-101 trp1-901 his3-Δ200 CHS5::3HASantos and Snyder (50Santos B. Snyder M. J. Cell Biol. 1997; 136: 95-110Crossref PubMed Scopus (171) Google Scholar)Y1306MATa ura3-52 lys2-801 ade2-101 trp1-901 his3-Δ200 CHS3::3HASantos and Snyder (50Santos B. Snyder M. J. Cell Biol. 1997; 136: 95-110Crossref PubMed Scopus (171) Google Scholar) Open table in a new tab Plasmid—Plasmids used in this study are listed in Table II. RCR1 (systematic name YBR005w) was amplified from genomic DNA by PCR using DNA polymerase Pyrobest (Takara). A BamHI site was placed at 219 bp upstream from the initiator methionine codon of RCR1, and a XhoI site was placed at 363 bp downstream from the stop codon (sense primer, 5′-CGGGATCCCGCCTCCTCTCTCGAAGC-3′; antisense primer, 5′-CCCTCGAGATCTATTGATCTCTGACGAGTA-3′). The PCR product was digested with BamHI and XhoI and ligated into pRS426 (21Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to generate pki72. To obtain the N-terminal myc epitope-tagged Rcr1 in yeast, the RCR1 open reading frame was amplified by PCR from pki72, placing a BamHI site at the start codon and SacI site at 363 bp downstream from the stop codon (sense primer, 5′-CGGGATCCATGGGACTTATTTCATACGAAAAT-3′; antisense primer, 5′-CGAGCTCATCTATTGATCTCTGACGAGTA-3′). The PCR product was digested with BamHI and SacI and ligated into pki80 (2μ, URA3, and a six myc-coding sequence) to generate pki94. The truncated Rcr1 constructs were made in the same way. The disruption plasmid pki106 (rcr1Δ::LEU2) was constructed by inserting the BamHI/PstI fragment obtained by PCR using the primers (sense primer, 5′-TGCACTGCAGAGGCATGGTAGATGCTATCG-3′; antisense primer, 5′-CGGGATCCTCCTTATTCTTAATTCGAGTTATA-3′) and the PstI/XhoI fragment obtained by PCR using the primers (sense primer, 5′-TGCACTGCAGATCTATTGATCTCTGACGAGTA-3′; antisense primer, 5′-CCCTCGAGACTGAATCTTTTTTTCCGGCTG-3′) in the BamHI/PstI and PstI/XhoI sites of pRS305, respectively. In this way, RCR1 was replaced by LEU2. Schemes for the other construction of the plasmids and the sequence of PCR primers are available upon request.Table IIPlasmids used in this studyPlasmidCharacteristicsReference/sourcepRS305LEU2Sikorski and Hieter (21Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)pRS4262μm, URA3Sikorski and Hieter (21Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)pki106rcr1Δ::LEU2This studymp34-82μm, URA3, expresses ERD1 from its own promoterThis studypki722μm, URA3, expresses RCR1 from its own promoterThis studypki942μm, URA3, expresses 6myc-RCR1 from SED5 promoterThis studypki1142μm, URA3, expresses 6myc-RCR1 (63-213 aa) from SED5 promoterThis studypki1152μm, URA3, expresses 6myc-RCR1 (69-213 aa) from SED5 promoterThis studypki1162μm, URA3, expresses 6myc-RCR1 (40-213 aa) from SED5 promoterThis studypki1262μm, URA3, expresses 6myc-RCR1 (1-160 aa) from SED5 promoterThis studypki1272μm, URA3, expresses 6myc-RCR1 (1-96 aa) from SED5 promoterThis studypki1282μm, URA3, expresses 6myc-RCR1 (1-70 aa) from SED5 promoterThis study Open table in a new tab Indirect Immunofluorescence Microscopy—Cells were prepared for indirect immunofluorescence microscopy as described previously (22Wooding S. Pelham H.R. Mol. Biol. Cell. 1998; 9: 2667-2680Crossref PubMed Scopus (158) Google Scholar). Log-phase cells grown at 30 °C were fixed by adding 2.5 ml of fresh 10% paraformaldehyde to 7.5 ml of yeast culture and pelleted by centrifugation. They were resuspended in 3.2 ml of PP (0.1 m potassium phosphate, pH 7.5), 1.8 ml of paraformaldehyde solution was added, and fixation was continued for an additional 15 min. Cells were then washed four times in PP and resuspended in 1 ml of SPP (PP with 1.2 m sorbitol) containing 100 mm dithiothreitol. 10 μl of lyticase was added, and incubation was continued at 30 °C. The spheroplasts were harvested by centrifugation and resuspended in 50 mm NH4Cl in SPP, and then in SPP before being transferred to polylysine-coated slides. The slides were immersed in methanol for 6 min and acetone for 30 s, both at -20 °C, and then air dried. Anti-myc mouse monoclonal antibody (9E10, Berkeley Antibody) and anti-Kar2 rabbit polyclonal antibody (a kind gift of Dr. Masao Tokunaga) were diluted to 1/40, 1/100 each in 1% skim milk, 0.1% bovine serum albumin, and 0.05% Tween 20 in Tris-buffered saline, and incubations were carried out overnight at 4 °C. Slides were washed and incubated in the secondary antibodies (fluorescein isothiocyanate-conjugated goat antibody to mouse immunoglobulin G and Texas Red-conjugated goat antibody to rabbit immunoglobulin G) for 2 h at room temperature, washed with phosphate-buffered saline, and mounted as described by Kilmartin and Adams (23Kilmartin J.V. Adams A.E. J. Cell Biol. 1984; 98: 922-933Crossref PubMed Scopus (549) Google Scholar). Images were obtained using an FV500 confocal laser-scanning microscope (Olympus, Tokyo, Japan). Biochemical Analyses—For preparation of yeast lysate, cells grown to log phase were harvested, washed, and resuspended in sorbitol buffer (1 m sorbitol, 1 mm MgCl2, 100 mm potassium phosphate, pH 7.5, 85 mm β-mercaptoethanol). Cells were converted to spheroplasts with lyticase (49Shen S.H. Chietien P. Bastien L. Slilaty S.N. J. Biol. Chem. 1991; 266: 1058-1063Abstract Full Text PDF PubMed Google Scholar) and homogenized in B88 (20 mm Hepes, 150 mm potassium acetate, 5 mm magnesium acetate, 200 mm sorbitol, pH 6.8) with protease inhibitors mixture (5 mm 1,10-phenanthroline, 2 μm pepstatin A, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin) and 1 mm phenylmethylsulfonyl fluoride (Wako Chemicals). Lysate was obtained after clearing centrifugation at 300 × g to remove unbroken cells. For subcellular fractionation, the cleared lysate was sequentially centrifuged to generate 10,000 × g pellet (P10), 100,000 × g pellet (P100), and 100,000 × g supernatant (S100) fractions. For solubilization, the lysate was treated on ice for 10 min with lysis buffer and lysis buffer containing either 1% Triton X-100, 0.1 m Na2CO3 (pH 11), or 2 m urea. Then a portion of the mixture was taken as the total fraction (T), and the remaining mixture was centrifuged at 100,000 × g and separated to the pellet (P) and the supernatant (S) fractions. Analyses of invertase, chitinase, and Gas1 protein were done as described in Hashimoto et al. (24Hashimoto H. Sakakibara A. Yamasaki M. Yoda K. J. Biol. Chem. 1997; 272: 16308-16314Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and Gentzsch and Tanner (25Gentzsch M. Tanner W. Glycobiology. 1997; 7: 481-486Crossref PubMed Scopus (141) Google Scholar). Protease Protection Assay—The KA31–1A spheroplasts harboring multicopy 6myc-RCR1 were prepared using lyticase in the presence of β-mercaptoethanol. Protease protection assays were performed as described previously (26Abeliovich H. Grote E. Novick P. Ferro-Novick S. J. Biol. Chem. 1998; 273: 11719-11727Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27Romano J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar), with the use of proteinase K (Roche Applied Science). Spheroplasts (10 optical density unit equivalents) were incubated with SPP, including combinations of proteinase K and/or 1% Triton X-100 for 5 min on ice. Reactions were terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mm. Bovine serum albumin was added as carrier (final concentration, 62.5 μg/ml) before proteins were precipitated with 10% trichloroacetic acid. Samples were resolved by SDS-PAGE and analyzed by immunoblotting for 6myc-Rcr1 or Kar2. Immunological Analyses—Immunoprecipitation was done as described previously (28Abe M. Noda Y. Adachi H. Yoda K. J. Cell Sci. 2004; 117: 5687-5696Crossref PubMed Scopus (29) Google Scholar). Western blots were probed with rabbit polyclonal antibodies against Kar2 or Gos1 and mouse monoclonal antibody against myc (9E10), HA 1The abbreviations used are: HA, hemagglutinin; ER, endoplasmic reticulum. (12CA5), or Pep12 (2C3-G4), followed by incubation with peroxidase-labeled goat antibody to rabbit IgG(H+L) and mouse IgG(H+L) (KPL, Gaithersburg, MD), respectively. Signals were detected by using a chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and Lumino-image analyzer (LAS-1000plus, Fujifilm, Tokyo, Japan). Quantitative β1,3-Glucan Measurement—Amount of β1,3-glucan per cell was measured using aniline blue as described previously (29Sekiya-Kawasaki M. Abe M. Saka A. Watanabe D. Kono K. Minemura-Asakawa M. Ishihara S. Watanabe T. Ohya Y. Genetics. 2002; 162: 663-676Crossref PubMed Google Scholar, 30Shedletzky E. Unger C. Delmer D.P. Anal. Biochem. 1997; 249: 88-93Crossref PubMed Scopus (85) Google Scholar) with some modification. In brief, cells were grown to A600 = 0.5 ∼ 0.8 and 2.5 × 106 cells were harvested. The cells were washed twice with TE (10 mm Tris·Cl, 1 mm EDTA, pH 8.0) and resuspended in 250 μl of TE. To the cells 6 n NaOH was added to a final concentration of 1 n, incubated at 80 °C for 30 min followed by an addition of 1.05 ml of AB mix (0.03% aniline blue (Wako), 0.18 n HCl, and 0.49 n glysine/NaOH, pH 9.5). The tube was vortexed briefly, then incubated at 50 °C for 30 min. Fluorescence of β-1,3-glucan was quantified using a spectrofluorometer (F-2500, Hitachi, Tokyo, Japan). The excitation wavelength was 400 nm, and the emission wavelength was 460 nm. Measurement of Chitin Content—Total cellular chitin was measured as described by Bulawa et al. (31Bulawa C.E. Slater M. Cabib E. Au-Young J. Sburlati A. Adair Jr., W.L. Robbins P.W. Cell. 1986; 46: 213-225Abstract Full Text PDF PubMed Scopus (181) Google Scholar) and outlined by Ketela et al. (32Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar) with some modification. In brief, washed cells (∼65–100 mg wet cells) were resuspended in 1 ml of 6% KOH and incubated at 80 °C for 90 min. After cooling at room temperature, 100 μl of glacial acetic acid was added. Insoluble material was washed twice with water and resuspended in 450 μl of 50 mm sodium phosphate (pH 6.3) containing 0.2 unit of Serratia marcescens chitinase (Sigma) and incubated at 37 °C for 2 h. After centrifugation, 400 μl of supernatant was incubated with 0.25 mg of Helix pomatia β-glucuronidase (Sigma) at 37 °C for 1 h, and then an aliquot of the mixture was assayed for N-acetylglucosamine content according to Reissig et al. (33Reissig J.L. Storminger J.L. Leloir L.F. J. Biol. Chem. 1955; 217: 959-966Abstract Full Text PDF PubMed Google Scholar). Calcofluor White Staining—Calcofluor white staining was performed as described previously (34Pringle J.R. Methods Enzymol. 1991; 194: 732-735Crossref PubMed Scopus (223) Google Scholar) with some modification. Briefly, log-phase cells grown at 30 °C were fixed with 3.7% paraformaldehyde and stained with 0.1 mg/ml Calcofluor white for 1 h. Cells were washed three times with phosphate-buffered saline and mounted on a slide in mounting medium (1 mg/ml p-phenylenediamine, phosphate-buffered saline, pH 9.0, 90% glycerol). Images were obtained using AX-80 microscope (Olympus). Identification of RCR1/YBR005w as a Multicopy Suppressor Gene of Congo Red Hypersensitivity of erd1Δ Mutant—ERD1 was previously found as a gene required for retention of the ER lumen proteins (35Pelham H.R. Hardwick K.G. Lewis M.J. EMBO J. 1988; 7: 1757-1762Crossref PubMed Scopus (189) Google Scholar), and its mutants show temperature-sensitive growth in YPD medium, Geneticin hypersensitivity, 2K. Imai, Y. Noda, H. Adachi, and K. Yoda, unpublished data. and glycosylation defect (36Hardwick K.G. Lewis M.J. Semenza J. Dean N. Pelham H.R. EMBO J. 1990; 9: 623-630Crossref PubMed Scopus (121) Google Scholar). We found that the erd1Δ mutant also shows hypersensitivity to Congo red and conducted a screening for multicopy suppressors that confer this to elucidate the function of the Erd1 protein. S. cerevisiae YKI59 (erd1Δ) was transformed with a multicopy library YNYL2 (URA3, 2μ). 82 out of 1,200,000 transformants formed colonies on SD medium containing 100 μg/ml Congo red. Seventy-six of them had ERD1. By sequencing and subcloning of the remaining six plasmids, we found that a previously uncharacterized open reading frame, YBR005w, was responsible for colony formation of YKI59 transformants on the SD plus Congo red medium (Fig. 1A). We named this gene RCR1 (resistance to Congo red 1). On the other hand, the other phenotype of erd1Δ, including the temperature sensitivity, glycosylation defect, and hypersensitivity to Geneticin, did not recover in the RCR1 transformant. Fig. 1B shows that no difference was found in N-glycosylation of invertase, O-glycosylation of chitinase, and maturation of glycosylphosphatidylinositol-anchored Gas1 protein. Furthermore, the Congo red resistance was not restricted in the erd1Δ mutant, and introduction of the multicopy RCR1 made the wild-type and various mutant yeast strains more resistant to the growth inhibitory action of Congo red (Fig. 1C). Introduction of the multicopy RCR1 also made these strains more resistant to Calcofluor white (Fig. 1D). Phenotypes of the rcr1Δ Null Mutant—Firstly, we examined the phenotype caused by an rcr1Δ null allele, because cells become significantly resistant to Congo red by the introduction of RCR1 in a CEN plasmid (data not shown). A heterologous diploid with RCR1/rcr1Δ was constructed from BY4743 and spores were dissected into tetrads. Sensitivity to SDS and growth at 37 °C were the same in the RCR1 and rcr1Δ haploid progenies. But the rcr1Δ progenies showed a higher sensitivity to 50 μg/ml Congo red or 40 μg/ml Calcofluor white in SD medium than the RCR1 progenies (Fig. 2A). The medium condition is important in determining the sensitivity, because all progenies grew similarly in YPD medium containing these compounds. Characterization of RCR1—RCR1 encodes a polypeptide of 213 amino acids with a calculated mass of 23.9 kDa. Another open reading frame YDR003w encoding a polypeptide of 210 amino acids has a sequence identity of 46% with Rcr1. We named this gene RCR2 (Fig. 3A). However, the multicopy RCR2 did not have an activity to confer Congo red resistance in any strain tested so far. The rcr2Δ null mutant did not show increased sensitivity to Congo red or Calcofluor white, and the rcr1Δ rcr2Δ double disruptant showed similar sensitivity to these dyes as the rcr1Δ single null mutant (Fig. 2A). These null alleles had no effect on glycosylation of invertase (Fig. 2B) and maturation of Gas1 protein (Fig. 2C). Therefore, it is unlikely that RCR1 and RCR2 have an essential redundant role in S. cerevisiae. The hydropathy plot suggests that Rcr1 and Rcr2 are integral membrane proteins with a single transmembrane domain (Fig. 3B) and the TMHMM program (available at www.cbs.dtu.dk/services/TMHMM/) predicts that amino acids 40–62 of Rcr1 form an α-helix that spans the membrane. It is noticeable that this transmembrane segment has an adjacent arginine-rich stretch (amino acids 64–71). The PEST find program (available at www.at.embnet.org/embnet/tools/bio/PESTfind/9) suggests that amino acids 99–114 is a region with a high PEST score (+6.84). The PEST regions are thought to endow several proteins with sensitivity to ubiquitination or proteasomal digestion. Localization of Rcr1 Protein—To make it clear that Rcr1 localizes in the membrane, we have first done a fractionation analysis. A 6myc epitope was added at the N terminus of Rcr1 for immunological detection, and the 6myc-Rcr1 protein was produced under the control of the SED5 promoter (pki94). By introduction of pki94, the yeast cells acquired a similar resistance to Congo red as the authentic RCR1 gene. The 6myc-Rcr1 protein migrated in SDS-PAGE to the position corresponding to a protein of 54 kDa, although its calculated molecular mass is about 34 kDa (data not shown). The spheroplasts of wild-type yeast having pki94 were lysed, and the lysate was subjected to 100,000 × g centrifugation. 6myc-Rcr1 was exclusively recovered in the pellet and not solubilized by treating with 0.1 m Na2CO3 or 2 m urea (Fig. 4A), but it was solubilized by 1% Triton X-100. These results indicate that Rcr1 is a typical integral membrane protein. Next, we examined the localization of Rcr1 by subcellular fractionation and immunofluorescent microscopy. A majority of 6myc-Rcr1 was recovered in P10 as well as the ER-marker protein Kar2, whereas a similar amount of the endosome-marker Pep12 or the Golgi-marker Gos1 was recovered equally in P10 and P100 (Fig. 4B). Immunofluorescent staining of myc epitope showed that 6myc-Rcr1 was present in a central ring and in peripheral ribbon-like structures beneath the cytoplasmic membrane. This immunological staining coincided well with that of the ER-marker Kar2 (Fig. 5). Topology of the Rcr1 Protein—To make a topological issue clear, the membrane fraction was treated with proteinase K and protection of the N-terminal 6myc tag was examined by immunoblotting. In the absence of a detergent, a 32-kDa remnant of 6myc-Rcr1 was detected suggesting that the C-terminal cytosolic region was digested (Fig. 6.). Kar2 protein in the lumen of the ER remained mostly intact. In the presence of a detergent, no myc signal was detected indicating the whole protein was digested and Kar2 became a smaller fragment that is intrinsically resistant to proteinase K digestion. When a tag was added to the C-terminal of Rcr1 to make Rcr1-6myc, no epitope signal was detected after proteinase digestion both in the presence and absence of detergent (data not shown). These results indicate that Rcr1 is a type I membrane protein with the N-terminal in the ER lumen and the C-terminal in the cytosol. Molecular Dissection of the Rcr1 Protein—To get clues to reveal the mechanism that makes the cell more resistant to Congo red, we dissected the Rcr1 molecule and sought to find which region is responsible for the activity. The truncated polypeptides at the N or C termini were produced by a multicopy plasmid under the SED5 promoter in an rcr1Δ strain of KA31 genetic background (Fig. 7A). A similar amount of polypeptide was produced because a similar intensity of myc signal at the N-terminal was detected in each construct by immunoblotting (data not shown). We found that the N-terminal luminal region is dispensable for Congo red resistance, because Rcr1 (amino acids 40–213) is active (Fig. 7B). The transmembrane domain is required for the activity, because Rcr1 (amino acids 63–213) and Rcr1 (amino acids 69–213) were inactive. A C-terminal region that has a low sequence similarity between Rcr1 and Rcr2 could be removed with a little decrease of activity and Rcr1 (amino acids 1–160) still had a significant activity. However, the cytosolic conserved region is essential, because Rcr1 (amino acids 1–96) and Rcr1 (amino acids 1–70) did not have an activity. A chimera protein consisting of amino acids 1–63 of Rcr1 and 64–210 of Rcr2 had activity (Fig. 7C), although Rcr2 itself did not confer Congo red resistance in the same expression construct. This suggested that the conserved region plays an important role for the activity of Rcr1. RCR2 may be a defective copy of a duplication of a common ancestor of the RCR1 gene." @default.
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• W2024253884 title "A Novel Endoplasmic Reticulum Membrane Protein Rcr1 Regulates Chitin Deposition in the Cell Wall of Saccharomyces cerevisiae" @default.
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