Title: Rad23 Provides a Link between the Png1 Deglycosylating Enzyme and the 26 S Proteasome in Yeast
Abstract: In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities. In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities. endoplasmic reticulum peptide:N-glycanase ubiquitin-like domain 5-fluoroorotic acid polyacrylamide gel electrophoresis green fluorescent protein hemagglutinin Proteins that transit through the secretory pathway are subjected to a quality control system (1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1066) Google Scholar) in the endoplasmic reticulum (ER)1 that recognizes aberrantly folded proteins/glycoproteins. It has been shown that in some cases these misfolded and/or unfolded proteins are degraded by ER-associated degradation mechanisms, which involves retrograde transfer of proteins from the ER to the cytosol followed by degradation by the proteasome (2Suzuki T. Yan Q. Lennarz W.J. J. Biol. Chem. 1998; 273: 10083-10086Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 3Brodsky J.L. McCracken A.A. Semin. Cell Dev. Biol. 1999; 10: 507-513Crossref PubMed Scopus (300) Google Scholar, 4Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 5Römisch K. J. Cell Sci. 1999; 112: 4185-4191Crossref PubMed Google Scholar, 6Johnson A.E. Haigh N.G. Cell. 2000; 102: 709-712Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Lord J.M. Davey J. Frigerio L. Roberts L.M. Semin. Cell Dev. Biol. 2000; 11: 159-164Crossref PubMed Scopus (39) Google Scholar). Previously, we described PNG1, a gene encoding a cytoplasmic deglycosylating enzyme, peptide:N-glycanase (PNGase), that is evolutionarily conserved throughout eukaryotes (8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar). It has been suggested that this enzyme activity is linked to a proteasomal degradation pathway and has a role in efficient degradation of glycoproteins by the proteasome (8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar, 9Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 10Wiertz E.J.H.J. Tortorella D. Bogyo M., Yu, J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (955) Google Scholar, 11Suzuki T. Kitajima K. Emori Y. Inoue Y. Inoue S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6244-6249Crossref PubMed Scopus (83) Google Scholar, 12Shamu C.E. Story C.M. Rapoport T.A. Ploegh H.L. J. Cell Biol. 1999; 147: 45-58Crossref PubMed Scopus (130) Google Scholar, 13Story C.M. Furman M.H. Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8516-8521Crossref PubMed Scopus (73) Google Scholar). This would be achieved by removing bulky N-linked glycans from misfolded glycoproteins that are translocated from the lumen of the ER into the cytosol for degradation. However, a physical link between the proteasome and this deglycosylating enzyme has not yet been described. Rad23p is known to have a pivotal role in nucleotide excision repair (14Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (257) Google Scholar, 15de Laar W.L. Jaspers N.G.L. Hoeijmakers J.H.J. Genes Dev. 1993; 13: 768-785Crossref Scopus (926) Google Scholar, 16Prakash S. Prakash L. Mutat. Res. 2000; 451: 13-24Crossref PubMed Scopus (286) Google Scholar). Yeast Rad23p stoichiometrically forms a complex with Rad4p to form nucleotide excision repair factor 2 (NEF2). Unlike other NER proteins, the biochemical functions of NEF2 still remain largely unknown. However, NEF2 was recently shown to bind specifically to damaged DNA in an ATP-independent manner (17Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 18Jansen L.E.T. Verlage A. Brouwer P. J. Biol. Chem. 1998; 273: 33111-33114Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Sugasawa K. Ng J.M. Masutani C. Iwai S. van der Spek P.J. Eker A.P. Hanaoka F Bootsma D. Hoeijmakers J.H. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar). While the absence of Rad4p (rad4Δ) causes extreme sensitivity to UV light in yeast, rad23Δ mutants exhibited only moderate UV sensitivity, indicating that Rad23p may affect the efficiency of the excision repair process rather than directly mediating the repair of damaged DNA (20Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 8385-8388Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 21Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 22Guzder S.N. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 8903-8910Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23Sugasawa K. Masutani C. Uchida A. Maekawa T. van der Spek P.J. Bootsma D. Hoeijmakers J.H. Hanaoka F. Mol. Cell. Biol. 1996; 16: 4852-4861Crossref PubMed Scopus (145) Google Scholar). One structural feature of Rad23p is that it contains a ubiquitin-like domain (UbL) at the N terminus that can bind to the 26 S proteasome (24Schauber C. Chen L. Tonganonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar). This association appears to be important for DNA repair (24Schauber C. Chen L. Tonganonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar, 25Watkins J.F. Sung P. Prakash L. Prakash S. Mol. Cell. Biol. 1993; 13: 7757-7765Crossref PubMed Scopus (217) Google Scholar, 26Russell S.J. Reed S.H. Huang W. Friedberg E.C. Johnston S.A. Mol. Cell. 1999; 3: 687-695Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In addition, Rad23p was shown to have an overlapping function with Rpn10p, a 19 S proteasome subunit that is known to be a multiubiquitin chain-binding receptor for the proteasome (27Lambertson D. Chen L. Madura K. Genetics. 1999; 153: 69-79Crossref PubMed Google Scholar). It has been suggested that the Rad23p is a negative regulator of multiubiquitin chain assembly (28Ortolan T.G. Tonhaokar P. Lambertson D. Chen L. Schauber C. Madura K. Nat. Cell Biol. 2000; 2: 601-608Crossref PubMed Scopus (161) Google Scholar). However, thus far, the link between DNA repair and proteasome degradation with respect to Rad23p function remains elusive. We report here a finding that the deglycosylation enzyme, Png1p, exists as a high molecular weight complex with Rad23p. The Png1p-Rad23p complex was found to be distinct from the well established DNA repair complex, Rad4p-Rad23p (NEF2). In addition, we report that the Png1p-Rad23p complex interacts with the 26 S proteasome. These findings led us to hypothesize that Rad23p may function to link the 26 S proteasome with other proteins, such as Rad4p or Png1p. This "escort" property of Rad23p may explain the complex effect of Rad23p-proteasome associations in a variety of cellular processes, including deglycosylation of glycoproteins slated for degradation. The yeast strains used in this study were the following: BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; Ref. 8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar); Research Genetic strain 10278 (BY4742 rad23Δ::KanMX4); L4O (MATa ade2 his3 leu2 trp1 LYS2::lexAop-HIS3 URA3::lexAop-lacZ; Ref. 29Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar); AMR70 (MATα ade2 leu2 his3 trp1 URA3::lexAop-lacZ; Ref. 29Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar); W303–1a (MATa ade2–101 his3–11, 15 leu2–3, 112 trp1–1 ura3–1 can1–100); TSY190 (W303–1arad23Δ::URA3 png1Δ::his5+(pombe)FOAR); and TSY195 (TSY190RPT1-GFP-HA::URA3::HIS3). TSY190 was prepared by crossing TSY146 (W303–1apng1Δ::his5+(pombe); Ref. 8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar) and MGSC101 (W303–1b (W303–1a MATα)rad23Δ::URA3; Ref. 30Verlage R.A. Zeeman A.-M. Lombaerts M. van de Putte P. Brouwer P. Mutat. Res. 1996; 362: 155-165Crossref PubMed Scopus (29) Google Scholar; kindly provided by Dr. Jaap Brouwer, Leiden) followed by isolating haploid segregants of the appropriate genotype and selection of 5-fluoroorotic acid (FOA)-resistant cells on FOA plates. TSY195 was prepared by transforming TSY190 with XhoI/NotI digests of pBS-CIM5-GFPHA-HU (Ref. 31Enenkel C. Lehmann A. Kloetzel P.-M. EMBO J. 1998; 17: 6144-6154Crossref PubMed Scopus (186) Google Scholar; kindly provided by Dr. Cordula Enenkel, Humboldt Universität, Berlin) and isolating Ura+His+ transformants. Correct integration of the transformant was confirmed by colony PCR as well as the expression of Rpt1-GFP-HAp by Western blotting using mouse anti-HA antibody (12CA5). Standard yeast media and genetic techniques were used (32Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar, 33Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar, 34Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar). PNGase activity was assayed in yeast lysates using fetuin-derived asialoglycopeptide I ([14C]CH3)2Leu-Asn(GlcNAc5Man3Gal3)-Asp-Ser-Arg) as described previously (35Suzuki T. Seko A. Kitajima K. Inoue Y. Inoue S. J. Biol. Chem. 1994; 269: 17611-17618Abstract Full Text PDF PubMed Google Scholar, 36Suzuki T. Park H. Kitajima K. Lennarz W.J. J. Biol. Chem. 1998; 273: 21526-21530Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Radioactivity was monitored on a PhosphorImager (Molecular Dynamics, Inc.) and quantitated using ImageQuant (version 1.2). One unit was defined as the amount of enzyme that catalyzed hydrolysis of 1 μmol of fetuin-derived asialoglycopeptide I/h. DNA manipulations were performed according to Sambrook et al. (37Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmids used in this study are listed in Table I. pCS13 was a kind gift from Dr. Kiran Madura. The yeast genes used in this study were isolated from the genomic DNA of W303-1a by polymerase chain reaction using Vent DNA polymerase (New England Biolabs). For isolation of the PNG1 gene, the following primers were used: 5′-AAAAAGAATTC-ATGGGAGAGGTATACGAAAAAA-3′ (5′-primer) and 5′-AAAAACTCGAG-CTATTTACCATCCTCCCCACGC-3′ (3′-primer). The amplified fragments were digested with EcoRI/XhoI and cloned into EcoRI/SalI sites of pBTM116 (29Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar) andEcoRI/XhoI sites of pRD53 (38Peter M. Neiman A.M. Park H.O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (193) Google Scholar). The pBTM116-PNG1 was subsequently digested with MluI/PstI and cloned into MluI/PstI sites of pBTM116-ADE2 (39Park H. Sternglanz R. Chromosoma ( Berl. ). 1998; 107: 211-215Crossref PubMed Scopus (20) Google Scholar) to give rise to pBTM116-ADE2-PNG1. pRD53-PNG1His6 was prepared by amplifying the PNG1 gene using another 3′-primer, 5′-AAAAACTCGAGTCAGTGGTGGTGGTGGTGGTGTTTACCATCCTCCCCACG-3′, and the EcoRI/XhoI fragments were cloned into pRD53. pRS314-GAL1PNG1His6 was prepared by digestion of pRD53-PNG1His6 withNotI/XhoI and cloning the PNG1-containing fragment into NotI/XhoI sites of pRS314 (40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). pESC-TRP-RAD4(Myc) was constructed by co-transformation ofApaI/SalI-digested pESC-TRP (Stratagene) and polymerase chain reaction-amplified RAD4 gene using the following primers: 5′-AGAAAAAACCCCGGATCCGTAATACGACTCACTATAGGGCGAATTCATGAATGAAGACCTGCCCAAGG-3′ (5′-primer) and 5′-AAGCTTACTCGAGGTCTTCTTCGGAAATCAACTTCTGTTCGTCGACGTCTGATTCCTCTGACATCTC-3′ (3′-primer). The Trp+ colonies were isolated, and the transformants bearing plasmids with the correct insert were identified by colony polymerase chain reaction. The expression of the Rad4Mycp was further confirmed by Western blotting. The RAD23 gene was isolated by polymerase chain reaction using the primers 5′-AAAAAGAATTCATGGTTAGCTTAACCTTTAAAAATTTC-3′ (5′-primer) and 5′-AAAAAGTCGACTCAGTCGGCATGATCGCTGAATAG-3′ (3′-primer), and the amplified DNA was digested with EcoRI/SalI and cloned into pGAD424 (41Chien C.-T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1224) Google Scholar). Other truncated versions of RAD23(amino acids 78–398, 186–398, 253–398, 1–185, 1–252, 1–317, and 1–354) were amplified in a similar manner using primers that containEcoRI and SalI sites and cloned into pGAD424. The sequences of the resulting constructs were confirmed.Table IPlasmids used in this studyPlasmids 1-aAll plasmids used contain the AmpR gene.DescriptionSourcepBTM116-PNG1PADH1::lexA-PNG1 TRP1 2μThis studypBTM116-ADE2-PNG1PADH1::lexA-PNG1 TRP1 ADE2 2μThis studypGAD424-RAD23PADH1::Gal4BD-RAD23 LEU2 2μThis studypGAD424-RAD23-(77–398)PADH1::Gal4BD-RAD23 77–398 LEU22μThis studypGAD424-RAD23-(146–398)PADH1::Gal4BD-RAD23 146–398 LEU22μThis studypGAD424-RAD23-(253–398)PADH1::Gal4BD-RAD23 253–398 LEU22μThis studypGAD424-RAD23-(1–354)PADH1::Gal4BD-RAD23 1–354 LEU22μThis studypGAD424-RAD23-(1–317)PADH1::Gal4BD-RAD23 1–317 LEU22μThis studypGAD424-RAD23-(1–252)PADH1::Gal4BD-RAD23 1–252 LEU22μThis studypCS13PCUP1::FLAG-RAD23 LEU22μRef. 27Lambertson D. Chen L. Madura K. Genetics. 1999; 153: 69-79Crossref PubMed Google ScholarpRD53URA3 CENRef. 38Peter M. Neiman A.M. Park H.O. van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (193) Google ScholarpRD53-PNG1PGAL1::PNG1 URA3 CENThis studypRD53-PNG1His6PGAL1::PNG1-His 6 URA3 CENThis studypRS314TRP1 CENRef.40Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpRS314-GAL1PNG1His6PGAL1::PNG1-His 6 TRP1 CENThis studyYEp351LEU2 2μRef.56Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1993; 2: 163-167Crossref Scopus (1083) Google ScholarpESC-TRP-RAD4(Myc)PGAL1::RAD4-Myc TRP1 2μThis study1-a All plasmids used contain the AmpR gene. Open table in a new tab The two-hybrid experiments were carried out as described previously (39Park H. Sternglanz R. Chromosoma ( Berl. ). 1998; 107: 211-215Crossref PubMed Scopus (20) Google Scholar). Strain L4O was transformed with pBTM116-ADE2-PNG1 (target plasmid) and a two-hybrid genomic library (pGAD library (Ref. 42James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar; kindly provided by Dr. Phillip James). After screening 2.2 × 106transformants in a yeast genomic library, two distinct GAD library plasmids that showed a reproducible His+β-galactosidase+ phenotype in PNG1-specific manner were recovered. These candidate GAD plasmids were then transformed into strain L4O and mated with AMR70 cells with each of several plasmids encoding various lexA-plasmids. These candidate plasmids did not exhibit an interaction with thelexA-lamin or lexA-SIR4 C terminus, both of which have been used to test for potential false positives in two-hybrid library screening (39Park H. Sternglanz R. Chromosoma ( Berl. ). 1998; 107: 211-215Crossref PubMed Scopus (20) Google Scholar). However, isolated plasmids showed the His+ β-galactosidase+ phenotype with the original target plasmids. Yeast cytosol was prepared using glass beads as described previously (43Rexach M.F. Schekman R.W. J. Cell Biol. 1991; 114: 219-229Crossref PubMed Scopus (229) Google Scholar) except that the extraction of cytosol was carried out in the presence of a protease inhibitor mixture (final concentrations: leupeptin, 1 μg/ml; antipain, 2 μg/ml; benzamide, 10 μg/ml; chymostatin, 1 μg/ml; pepstatin, 1 μg/ml; phenylmethanesulfonyl fluoride, 1 mm). Preparation of a crude extract of bacterially expressed Png1p was reported elsewhere (8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar). 0.5 ml (∼10 mg of protein) of cytosol were loaded on a Sephacryl S-300 column (Amersham Pharmacia Biotech; 1.5 × 50 cm) equilibrated with elution buffer (20 mm Hepes buffer (pH 6.8), 5 mm magnesium acetate, 1 mmdithiothreitol, 2 mm ATP, 150 mm NaCl, and 0.4 m sorbitol with protease inhibitor mixture as described above), and fractions of 0.9 ml were collected. Fractions were assayed for PNGase activity. For protein determination, 0.3 ml of fractions were precipitated with 10% trichloroacetic acid, and the tagged protein was visualized by Western blotting. Western blot analysis was carried out as described (8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar) using 1:10 dilution of mouse anti-HA (tissue culture supernatant; 12CA5) or 1:1000 dilution of rabbit or mouse anti-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) rabbit or mouse anti-His6 (Santa Cruz Biotechnology), or mouse anti-FLAG antibody (M2; Sigma) followed by a 1:2000 dilution with the anti-rabbit or mouse IgG horseradish peroxidase-conjugated secondary antibody (Roche Molecular Biochemicals). 10% SDS-PAGE gels were used, and gels were visualized using chemiluminescence (KPL) after exposure to medical x-ray film (Fuji). Immunoprecipitation experiments were carried out as previously described (27Lambertson D. Chen L. Madura K. Genetics. 1999; 153: 69-79Crossref PubMed Google Scholar). Briefly, cell extracts were prepared in lysis buffer (20 mm Hepes-KOH, pH 7.5, 100 mm potassium acetate, 5 mm EDTA, 10% glycerol) including various protease inhibitors as described above, and equal amounts of extract (2 mg of total protein) were incubated with 20 μl of protein G-agarose with and without respective anti-Tag antibodies (rabbit anti-Myc (Santa Cruz Biotechnology); rabbit anti-HA (Santa Cruz Biotechnology); rabbit anti-His6 (Santa Cruz Biotechnology), 1:50 in dilution; or mouse anti-FLAG antibody (Sigma), 1:250 in dilution) and incubated overnight at 4 °C. The immunoprecipitates were washed twice with buffer A (50 mmHepes-NaOH, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100), dissolved in 20 μl of sample buffer, and analyzed by 10% SDS-PAGE. Subsequently, Western blotting analysis was carried out as described above. For qualitative UV sensitivity analysis, cells were grown in synthetic dextrose (SD) −Leu −Ura +galactose or −Leu −Ura +glucose medium to saturation. Cells were further cultured in the presence of 0.1 mmCuSO4 for another 2 h, and the cell density was normalized to A 600 = 1.0 (∼5.0 × 106 cells/ml). Cells were taken from the aliquot with Q-tips and streaked as a single line onto the respective plate (−Leu −Ura +galactose +0.1 mm CuSO4 or −Leu −Ura +glucose +0.1 mm CuSO4). After these plates were covered with a glass plate, they were placed 20 cm distant from the germicidal UV light (254 nm; Sylvania). Then the glass plate was slid parallel to the line of cells so that the cells were exposed to UV for different times from 0 to 8 s. The plate was then incubated at 25 °C in the dark for 4 days. If a cell line had UV sensitivity, cells would not be expected to grow all the way across the line when exposed to UV. In contrast, wild-type cells would be expected to exhibit growth across the entire line even when they were exposed to the maximum dose of UV. For quantitative assay for UV treatment, cells were grown in the respective medium (SD −Leu −Ura +glucose or SD −Leu −Ura +galactose) overnight, and after 0.1 mm CuSO4was added, they were cultured for 2 h, plated on YPAD at appropriate dilutions, and then exposed to 254-nm UV light using a UV cross-linker (UV Statalinker model 1800; Stratagene) at given doses. Cells were plated in triplicate and incubated at 25 °C in the dark for 3 days, and the number of surviving colonies were counted. Our initial assumption that Png1p might be a part of a multiprotein complex in yeast was based on the results of gel filtration analysis of a cell-free yeast extract. As shown in Fig.1, PNGase activity measurement of the gel filtration fractions showed distinct differences between the elution position of Png1p in the yeast cytosol and that of Png1p expressed inEscherichia coli. While the bacterially expressed protein showed the expected molecular mass of a monomer form (∼45 kDa), the yeast cytosol protein had a much higher molecular mass (estimated to be ∼200 kDa). This result suggested that in the yeast cytosol Png1p may bind to other proteins to form a high molecular weight protein complex. The observation described above led us to carry out two-hybrid library screening withPNG1 as a bait to search for possible Png1p-binding proteins. Upon screening 2.2 × 106 transformants with a yeast genomic library, we recovered two distinct plasmids that showed a reproducible His+ β-galactosidase+phenotype in a target plasmid (lexA-PNG1)-specific manner. Sequencing of inserts recovered from these two plasmids showed that they consisted of two different Rad23p fragments (one containing amino acid residues 218–398 and the other containing residues 229–398) fused in frame to the GAL4 activation domain sequence (Fig.2). These plasmids did not exhibit an interaction with either lexA-lamin or lexA-SIR4 C terminus, which have been used to test for potential false positives in two-hybrid library screening (39Park H. Sternglanz R. Chromosoma ( Berl. ). 1998; 107: 211-215Crossref PubMed Scopus (20) Google Scholar), suggesting that the interaction observed was specific (data not shown). To confirm the interaction of Png1p with Rad23p in vivo, gel filtration analysis was carried out to examine the migration of these two proteins. First, we determined the elution position of Png1p inrad23Δ cells. As shown in Fig.3, a drastic shift in elution position of this enzyme to a lower apparent mass was observed inrad23Δ cells, and the elution position was similar to that for bacterially expressed Png1p. This result suggested that the high molecular complex containing Png1p might also contain Rad23p. To provide direct evidence for interaction of Rad23p and Png1p, FLAG-tagged Rad23p (FLAG-Rad23p; Ref. 24Schauber C. Chen L. Tonganonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar) was expressed under an inducible CUP1 promoter. This protein construct was used to show interaction of Rad23p with the 26 S proteasome as well as with Rad4p (24Schauber C. Chen L. Tonganonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar, 27Lambertson D. Chen L. Madura K. Genetics. 1999; 153: 69-79Crossref PubMed Google Scholar). Png1p is known to be present in extremely low abundance in cells under normal experimental conditions (8Suzuki T. Park H. Hollingsworth N.M. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1051Crossref PubMed Scopus (190) Google Scholar), while Rad23p is relatively abundant (17Guzder S.N. Bailly V. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1998; 273: 31541-31546Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Therefore, only a minute fraction of Rad23p was expected to be bound to Png1p; this would make it difficult to observe co-migration of Png1p and Rad23p. For this reason, Png1p was overexpressed using a inducible GAL1 promoter in apng1Δ rad23Δ strain for this experiment (TSY190; for details of genotypes of strains, see "Experimental Procedures"). When both proteins were expressed, Png1p exhibited two peaks of activity, one corresponding to the higher molecular mass peak and one similar to bacterially expressed Png1p (Fig.4 A). FLAG-Rad23p was shown to co-migrate with the first peak and not with the second peak (Fig. 4,A and B). The proteasome was shown to be present in fractions 34–40 by activity assay as well as Western blotting using anti-Rpt1p antibody, showing that the first PNGase activity peak did not contain the proteasome complex (data not shown). When only Png1p was overexpressed in the absence of Rad23p (rad23Δ), the higher molecular weight peak was not detectable (data not shown; see also Fig. 3). When only Rad23p was overexpressed in the absence of Png1p (png1Δ), the elution position of Rad23p was shifted to a lower molecular weight (Fig. 4 C), suggesting that when Png1p was expressed, all of Rad23p detected co-migrated with Png1p. Having biochemical evidence that Png1p binds to Rad23p in yeast cytosol, we determined which domain of Rad23p was involved in interaction with Png1p using the two-hybrid assay. Deletion constructs of GAL4 activation domain-RAD23 were tested for binding against lexA-PNG1. As shown in TableII, deletion of N-terminal portions of Rad23p did not have a significant effect on its binding to Png1p. In sharp contrast, when the C-terminal region of Rad23p was truncated, no interaction with Png1p was observed. This result most likely suggested that the C-terminal ubiquitin-associated domain (UBA) of Rad23p (see Fig. 2) was important for its binding to Png1p.Table IIMapping of interaction domains of the Rad23p to Png1p assessed by yeast two-hybrid assaypGAD424 constructsGrowth on SD minus histidine plateβ-Galactosidase assay 2-aNo color change on β-galactosidase assay; number of plus signs indicates the intensity of color on β-galactosidase assay.Rad23p-(1–398) (full-length)+++Rad23p-(77–398) (minus N terminus)++++Rad23p-(186–398)++++Rad23p-(218–398) 2-bThis plasmid was originally isolated from the pGAD library.++++Rad23p-(253–398)++Rad23p-(1–354) (minus C terminus)−−Rad23p-(1–317)−−Rad23p-(1–252)−−2-a No color change on β-galactosidase assay; number of plus signs indicates the intensity of color on β-galactosidase assay.2-b This plasmid was originally isolated from the pGAD library. Open table in a new tab Since Rad23p has been shown to require the N-terminal UbL for its interaction with the 26 S proteasome (24Schauber C. Chen L. Tonganonkar P. Vega I. Lambertson D. Potts W. Madura K. Nature. 1998; 391: 715-718Crossref PubMed Scopus (408) Google Scholar), we assumed that Png1p might associate with the 26 S proteasome through Rad23p. To test this hypothesis, co-immunoprecipitation analysis with His6-tagged Png1p (Png1-His6p) and the GFP/HA-tagged 26 S proteasome subunit, Rpt1p, was carried out. The His6-tagged Png1p was expressed under the GAL1 promoter. The His6-tagged Png1p was shown to express enzyme activity (data not shown). The GFP/HA-tagged Rpt1p was previously shown to be integrated into a functional 26 S proteasome complex (31Enenkel C. Lehmann A. Kloetzel P.-M. EMBO J. 1998; 17: 6144-6154Crossref PubMed Scopus (186) Google Scholar). Cell lysates were prepared from cells expressing Png1-His6p with or without FLAG-Rad23p in png1Δ rad23Δ cells, and co-immunoprecipitation was carried out using anti-His6 or anti-HA antibody. As shown in Fig. 5, Png1p and Rpt1p co-immunoprecipitated considerably more than background level, which might represent nonspecific interaction of proteins with resin (compare lanes 1 and 3 withlanes 2 and 4). This interaction was observed only in the presence of Rad23p (Fig. 5; lanes 5 and 7). The fraction of Png1p-Rad23p bound to the 26 S proteasome under these experimental conditions was estimated to be 2–5% based on a comparison of intensity of a band detected by Western blotting of whole extract with the intensity of immunoprecipitate pulled down by Rpt1-GFP-HAp (data not shown). The C-terminal domain of Rad23p, identified above as a critical binding region to Png1p, was previously shown to be critical for the Rad23p-Rad4p interactions (44Wang Z. Wei S. Reed S.H. Wu X. Svejtrup J.Q. Feaver W.J. Kornberg R.D. Friedberg E.C. Mol. Cell. Biol. 1997; 17: 635-643Crossref PubMed Scopus (67) Google Scholar). Therefore, it was possible that Png1p and Rad4p might