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• W4234773207 abstract "In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination. In this work, we determine that the Saccharomyces cerevisiae Ccr4-Not complex controls ubiquitination of the conserved ribosome-associated heterodimeric EGD (enhancer of Gal4p DNA binding) complex, which consists of the Egd1p and Egd2p subunits in yeast and is named NAC (nascent polypeptide-associated complex) in mammals. We show that the EGD complex subunits are ubiquitinated proteins, whose ubiquitination status is regulated during cell growth. Egd2p has a UBA domain that is not essential for interaction with Egd1p but is required for stability of Egd2p and Egd1p. Ubiquitination of Egd1p requires Not4p. Ubiquitination of Egd2p also requires Not4p, an intact Not4p RING finger domain, and all other subunits of the Ccr4-Not complex tested. In the absence of Not4p, Egd2p mislocalizes to punctuate structures. Finally, the EGD complex can be ubiquitinated in vitro by Not4p and Ubc4p, one of the E2 enzymes with which Not4p can interact. Taken together our results reveal that the EGD ribosome-associated complex is ubiquitinated in a regulated manner, and they show a new role for the Ccr4-Not complex in this ubiquitination. The yeast Ccr4-Not is a multifunctional complex composed of nine identified subunits (NotI-5p, Ccr4p, Caf1p, Caf40p, and Caf130p). It exists in at least two forms of 1.2 and 2 MDa that are conserved across the eukaryotic kingdom (for reviews, see Refs. 1Collart M.A. Gene (Amst.). 2003; 313: 1-16Crossref PubMed Scopus (154) Google Scholar, 2Collart M.A. Timmers H.T. Prog. Nucleic Acids Res. 2004; 77: 289-322Crossref PubMed Scopus (107) Google Scholar, 3Denis C.D. Chen J. Nucleic Acids Res. Mol. Biol. 2003; 73: 221-250Crossref PubMed Scopus (121) Google Scholar). The complex is involved in several different cellular pathways. First, it regulates transcription, and controls the appropriate distribution of TFIID across promoters (4Lenssen E. James N. Pedruzzi I. Dubouloz F. Cameroni E. Bisig R. Maillet L. Werner M. Roosen J. Petrovic K. Winderickx J. Collart M.A. De Virgilio C. Mol. Cell. Biol. 2004; 25: 488-498Crossref Scopus (57) Google Scholar). Second, Ccr4p and Caf1p are the major deadenylase in Saccharomyces cerevisiae, and play a role in mRNA degradation (5Tucker M. Staples R.R. Valencia-Sanchez M.A. Muhlrad D. Parker R. EMBO J. 2002; 21: 1427-1436Crossref PubMed Scopus (266) Google Scholar). Third, the Ccr4-Not complex controls the post-transcriptional modification and activity of the environmental stress transcription factor Msn2p (4Lenssen E. James N. Pedruzzi I. Dubouloz F. Cameroni E. Bisig R. Maillet L. Werner M. Roosen J. Petrovic K. Winderickx J. Collart M.A. De Virgilio C. Mol. Cell. Biol. 2004; 25: 488-498Crossref Scopus (57) Google Scholar, 6Lenssen E. Oberholzer U. Labarre J. de Virgilio C. Collart M.A. Mol. Microbiol. 2002; 43: 1023-1037Crossref PubMed Scopus (63) Google Scholar). Taken together, the studies points to both cytoplasmic and nuclear roles for the Ccr4-Not complex, and both cytoplasmic and nuclear localizations for subunits of this complex have been described (7Collart M.A. Struhl K. EMBO J. 1993; 12: 177-186Crossref PubMed Scopus (87) Google Scholar, 8Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). The precise role of the Ccr4-Not complex has not been determined, but our current model is that it serves as a platform that regulates several different cellular functions in response to changes in environmental signals, such as glucose depletion (for review, see Refs. 1Collart M.A. Gene (Amst.). 2003; 313: 1-16Crossref PubMed Scopus (154) Google Scholar and 2Collart M.A. Timmers H.T. Prog. Nucleic Acids Res. 2004; 77: 289-322Crossref PubMed Scopus (107) Google Scholar). We have little knowledge about the specific function of the individual subunits. Not4p has a conserved N-terminal domain that contains an atypical C4C4 type RING finger (9Hanzawa H. de Ruwe M.J. Albert T.K. Van de Vliet P.C. Timmers M.H.T. Boelens R. J. Biol. Chem. 2001; 276: 10185-10190Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), a zinc-binding domain that defines a large family of E3 ubiquitin ligases (for review, see Ref. 10Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1021) Google Scholar). Consistent with a role of Not4p as an E3 ligase, several E2 enzymes (UBCH6, UBCH9, and UBCH5b) were isolated in a two-hybrid selection for partners of the human CNOT4 (11Albert T.K. Hanzawa H. Legtenberg Y.I. deRuwe M.J. van der Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar). CNOT4 was subsequently shown to be capable of auto-ubiquitination in vitro, and to require its RING finger to functionally complement the deletion of NOT4 in yeast (11Albert T.K. Hanzawa H. Legtenberg Y.I. deRuwe M.J. van der Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar). Yeast Ubc4p and Ubc5p are homologous to the human E2 proteins identified as partners for CNOT4, and they interact with Not4p in the two-hybrid assay (11Albert T.K. Hanzawa H. Legtenberg Y.I. deRuwe M.J. van der Heuvel F.A. Collart M.A. Boelens R. Timmers H.T. EMBO J. 2002; 21: 355-364Crossref PubMed Scopus (158) Google Scholar, 12Winkler G.S. Albert T.K. Dominguez C. Legtenberg Y.I. Boelens R. Timmers H.T. J. Mol. Biol. 2004; 12: 157-165Crossref Scopus (86) Google Scholar). These yeast E2 enzymes can work with many E3 enzymes and have been associated with diverse cellular functions including the stress response, the degradation of short-lived proteins, endocytosis, and trafficking of membrane proteins and finally the degradation of cotranslationally damaged proteins (13Chuang S.M. Madura K. Genetics. 2005; 171: 1477-1484Crossref PubMed Scopus (29) Google Scholar, 14Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (407) Google Scholar, 15Kiel J.A.K.W. Emmrich K. Meyer H.E. Kunau W.-H. J. Biol. Chem. 2005; 280: 1921-1930Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16Kus B.M. Caldon C.E. Andorn-Broza R. Edwards A.M. Proteins Struct. Funct. Bioinf. 2004; 54: 455-467Crossref PubMed Scopus (63) Google Scholar, 17Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 18Horak J. Biochim. Biophys. Acta. 2003; 1614: 139-155Crossref PubMed Scopus (63) Google Scholar). In this work, we determine that the Ccr4-Not complex contributes to the ubiquitination and regulation of the conserved enhancer of Gal4p DNA binding (EGD) 2The abbreviations used are: EGD, enhancer of Gal4p DNA binding; NAC, nascent polypeptide-associated complex; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; SB, sample buffer; TE, total extract; GST, glutathione S-transferase; HA, hemagglutinin. 2The abbreviations used are: EGD, enhancer of Gal4p DNA binding; NAC, nascent polypeptide-associated complex; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; SB, sample buffer; TE, total extract; GST, glutathione S-transferase; HA, hemagglutinin. complex, which is named nascent polypeptide-associated complex (NAC) in mammals, and consists of the Egd1p (and its Btt1p homolog expressed at much lower levels (1/100) (19Rieimann B. Bradsher J. Franke J. Hartmann E. Wiedmann M. Prehn S. Wiedmann B. Yeast. 1999; 15: 397-407Crossref PubMed Scopus (71) Google Scholar)) (βNAC) and Egd2p (αNAC) subunits in yeast. Both subunits contain similar NAC domains through which they can dimerize and Egd2p additionally contains a ubiquitin-binding domain UBA (20Spreter T. Pech M. Beatrix B. J. Biol. Chem. 2005; 280: 15849-15854Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) at its C terminus. The Egd1p/Egd2p heterodimer is thought to act as a dynamic component of the ribosomal exit tunnel, protecting the emerging polypeptides from interaction with other cytoplasmic proteins to ensure appropriate nascent protein targeting (for review, see Ref. 22Rospert S. Dubaquie´ Y. Gautschi M. Cell. Mol. Life Sci. 2002; 59: 1632-1639Crossref PubMed Scopus (143) Google Scholar). Recently, it was found that the ribosomal protein L25 (encoded by RPL25) is the docking site for the EGD complex to ribosomes, in close proximity to the ribosome exit tunnel (23Grallath S. Schwarz J.P. Bottcher U.M. Bracher A. Hartl F.U. Siegers K. EMBO Rep. 2006; 7: 78-84Crossref PubMed Scopus (21) Google Scholar, 24Wegrzyn R.D. Hofmann D. Merz F. Nikolay R. Rauch T. Graf C. Deuerling E. J. Biol. Chem. 2006; 281: 2847-2857Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the EGD complex has also been associated with transcription regulation (25Parthun M.R. Mangus D.A. Jaehning J.A. Mol. Cell. Biol. 1992; 12: 5683-5689Crossref PubMed Scopus (40) Google Scholar, 26Moncollin V. Miyamoto N.G. Zheng X.M. Egly J.-M. EMBO J. 1986; 5: 2577-2584Crossref PubMed Scopus (68) Google Scholar, 27Zheng X.M. Black D. Chambon P. Egly J.M. Nature. 1990; 344: 556-559Crossref PubMed Scopus (96) Google Scholar, 28Zheng X.M. Moncollin V. Egly J.M. Chambon P. Cell. 1987; 50: 361-368Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 29Que´lo I. Gauthier C. Hannigan G.E. Dedhar S. St-Arnaud R. J. Biol. Chem. 2004; 279: 43893-43899Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), mostly in situations of unequal expression of either EGD subunit. It could be that the EGD complex performs both a cytoplasmic and a nuclear function, but its exact function in vivo remains to be elucidated. We show that both Egd1p and Egd2p are ubiquitinated proteins whose ubiquitination status changes during cell growth, and we find that the ubiquitination of Egd1p and Egd2p is dependent on Not4p and the Ccr4-Not complex. We also demonstrate in vitro ubiquitination of Egd2p by Not4p and its interacting E2 enzyme Ubc4p. Finally, we find that in the absence of Not4p, Egd2p mis-localizes. Our results present the first evidence for a role of Not4p in protein ubiquitination in vivo, underline the importance of the Ccr4-Not complex for this function of Not4p, and identify a substrate for this E3 ligase. Finally our results show that Not4p contributes to the appropriate localization of its substrate in vivo. Media and Strains—All media were standard. The strains used in this work derive from MY1 (30Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (171) Google Scholar) (Table 1). Single-step deletions and/or tagging of genes were performed by PCR according to Ref. 31Longtine M.S. McKenzie A.R. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4160) Google Scholar. Strain MY4448 was performed by amplifying the sequence of Egd2 between nucleotides 1 and 407. Strain MY4514 was performed by amplifying NOT4 sequences harboring the point mutation 105CTT-GCT105 (Not4L35Ap) by PCR, cloning the mutant allele of NOT4 in a URA3 integrative vector, and integration of the mutant allele at the NOT4 locus to replace endogenous NOT4.TABLE 1Yeast strains used in this studyStrainGenotypeSourceMY1MATa ura3-52 trp1 leu2::PET56 gcn4Ref. 30Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (171) Google ScholarYOU584MATa ura3-52 trp1 leu2::PET56 gcn4 not4::LEU2Ref. 6Lenssen E. Oberholzer U. Labarre J. de Virgilio C. Collart M.A. Mol. Microbiol. 2002; 43: 1023-1037Crossref PubMed Scopus (63) Google ScholarMY1719MATa ura3-52 trp1 his3 leu2::PET56 gcn4 not5::LEU2This workMY2184MATa ura3-52 trp1 leu2::PET56 gcn4 not2::KanMX4This workMY3593MATa ura3-52 trp1 leu2::PET56 gcn4 not4::KanMX4This workMY3608MATa ura3-52 trp1 leu2::PET56 gcn4 not3::KanMX4This workMY3609MATa ura3-52 trp1 leu2::PET56 gcn4 egd2::KanMX4This workMY3610MATa ura3-52 trp1 leu2::PET56 gcn4 egd1::KanMX4This workMY3611MATa ura3-52 trp1 leu2::PET56 gcn4 btt1::KanMX4This workMY3612MATa ura3-52 trp1 leu2::PET56 gcn4 egd1::EGD1-HA3-KanMX4This workMY3623MATa ura3-52 trp1 leu2::PET56 gcn4 not4::LEU2 egd2::KanMX4This workMY3624MATa ura3-52 trp1 leu2::PET56 gcn4 not4::LEU2 egd1::EGD1-HA3-KanMX4This workMY4417MATa ura3 trp1 his3 LEU2::LexAOp6-LEU2 pJK103 egd2::NatMX2This workMY4446MATa ura3-52 trp1 leu2::PET56 gcn4 caf40::CAF40-GST-TRP1This workMY4448MATa ura3-52 trp1 leu2::PET56 gcn4 egd2::EGD 2DUBA-TRP1This workMY4491MATa ura3-52 trp1 leu2::PET56 gcn4 not4::KanMX4 egd1::EGD1-HA3-KanMX4This workMY4514MATa ura3-52 trp1 leu2::PET56 gcn4 not4::not4-L35A-URA3-KanMX4 egd1::EGD1-HA3-KanMX4This workMY4519MATa ura3-52 trp1 leu2::PET56 gcn4 egd1::KanMX4This workEGY48MATa ura3 trp1 his3 LEU2::LexAOp6-LEU2 pJK103Ref. 38Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (664) Google Scholar Open table in a new tab DNA Constructs—pLex202-EGD1 and pJG4–5-EGD1 were created by subcloning an EcoRI-NheI fragment from pQE30-EGD1 (kind gift from Elke Deuerling) into yEPlac112 between EcoRI and XbaI, leading to pMAC518. Then, an EcoRI-SalI fragment from pMAC518 was cloned into pLex202 or pJG4–5, leading to pMAC519 and pMAC520. pLex202-EGD2 and pJG4–5-EGD2 were created by subcloning an EcoRI-SalI fragment from pQE31-EGD2 (kind gift from Elke Deuerling) into pLex202 or pJG4–5 leading to pMAC521 and pMAC520, respectively. pJG4–5-UBA and pJG4–5-EGD2ΔUBA were created by amplifying the UBA domain of Egd2p (nucleotides 407 to 525) and EGD2ΔUBA (nucleotides 1 to 408) by PCR, and cloning into pJG4–5 between BamHI and SalI, leading to pMAC550 and pMAC551. pADH-GFP-EGD2 with or without an additional histidine tag was made by cloning a MunI-SalI or EcoRI-SalI fragment from pQE31-EGD2 into a pRS414-derived plasmid expressing GFP from the ADH1 promoter (pMAC392) between the EcoRI and XhoI sites. To make pADH-GFP-EGD2ΔUBA we cloned the EcoRI-EcoRV EGD2 fragment from pQE31-EGD2 into pBSSK between EcoRI and HincII, and the EcoRI-XhoI fragments from this clone were subsequently cloned into pMAC392. To construct a plasmid expressing histidine-tagged Egd1p, EGD1 sequences amplified by PCR were cloned between SacI and SpeI of pMAC334 (32Creton S. Svejstrup J. Collart M.A. Genes Dev. 2002; 16: 3265-3276Crossref PubMed Scopus (22) Google Scholar) leading to pMAC538. Two-hybrid Assays—The two-hybrid assays were performed as already described in previous studies (30Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (171) Google Scholar). β-Galactosidase activity was revealed by overlaying transformants with 10 ml of 0.5% agar, 0.1 m sodium phosphate buffer, pH 7.0, 0.4 mg/ml X-gal and incubating the plates for 1–24 h at 30 °C. To detect expression of bait and prey, proteins were extracted from transformants grown in 2% galactose up to an A600 of 0.6 using the post-alkaline method (33Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (669) Google Scholar). Recombinant Protein Purification—His6-Ubc4p (from pQE-32-NOT4; Qiagen, kind gift from Klaas Mulder) and His6-Not4p (from pET-15b, Novagen) were expressed and purified as described (9Hanzawa H. de Ruwe M.J. Albert T.K. Van de Vliet P.C. Timmers M.H.T. Boelens R. J. Biol. Chem. 2001; 276: 10185-10190Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). His6-Egd1p (from pQE-30-EGD1, Qiagen) and His6-Egd2p (from pQE-31-EGD2, Qiagen) were expressed separately or co-expressed (from pQE-31-EGD2-EGD1, Qiagen, kind gift from Elke Deuerling) in MH1 cells in the presence of pRARE (Novagen) and purified as described (9Hanzawa H. de Ruwe M.J. Albert T.K. Van de Vliet P.C. Timmers M.H.T. Boelens R. J. Biol. Chem. 2001; 276: 10185-10190Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Affinity Purification—For purification of the EGD complex using the strain expressing histidine-tagged Egd1p, 180 A600 units of cells were collected in exponential phase, resuspended in 3 ml of buffer B (50 mm Tris-HCl, pH 7.0, 20 mm imidazole, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 mm 2-mercaptoethanol) with 50 mm NaCl (B50) and broken during 5 min at 4 °C with 2.4 ml of glass beads. After a 20-min centrifugation of 16,000 × g at 4 °C, protein extracts were incubated with 120 μl of nickel-nitrilotriacetic acid-agarose (Qiagen) during 2 h at 4 °C, washed 3 times with 1 ml of B50 buffer and 3 times with 1 ml of B100 buffer (buffer B with 100 mm NaCl). Proteins were eluted with 60 μl of 2× Laemmli sample buffer (SB) (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar). 20 μl of the samples were loaded on SDS gels and further analyzed by Western blotting. For purification of the Ccr4-Not complex, cells expressing Caf40p-GSTp from 2 liters of culture in exponential phase were broken using FastPrep in buffer A (20 mm Tris, pH 8, 100 mm potassium acetate, 5 mm MgCl2, 10% glycerol). After ultracentrifugation (100,000 × g, 1 h) the total extract (TE) was loaded on a 1 ml of GST-trap HP column, the column was washed with 5 ml of buffer A, and proteins were eluted with 5 ml of buffer A containing 20 mm reduced glutathione. The eluted proteins were loaded on a MonoQ PC 1.6/5 column and eluted with a linear gradient of potassium acetate (0–1.5 m) on buffer A. 50 μl of the fractions were collected and analyzed with SDS-PAGE and Western blot. Stability Assay—Cycloheximide (30 μg/ml final concentration) was added to cells growing exponentially for 24 h (A600 of 0.5). Proteins were extracted by post-alkaline lysis and 5–10 μl of the samples was analyzed with SDS-PAGE and Western blot. In Vivo Ubiquitination Assay—Cells expressing His6-ubiquitin under the control of a copper-dependent promoter were grown in medium containing 0.1 mm CuSO4 and 50 A600 units were collected at different times of growth. Cell pellets were weighed and resuspended in G-buffer (100 mm sodium Pi, pH 8.0, 10 mm Tris-HCl, 6 m guanidium chloride, 5 mm imidazole, 0.1% Triton X-100) to 50 mg/ml. 1 ml of cell suspension was disrupted with 0.6 ml of glass beads during 6 min at 4 °C and spun for 20 min at 13,000 × g. To remove guanidium chloride, 20 μl of the supernatants were diluted in 1.2 ml of water and concentrated with Strataclean resin (Stratagene) and eluted with 50 μl of Laemmli SB. 3–5 μl of TE were analyzed by Western blot with the relevant antibodies. The rest of the supernatant was incubated with 30 μl of nickel-nitrilotriacetic acid-agarose (Qiagen) for 2 h at room temperature with mild rotation. The agarose beads were washed 3 times with 0.5 ml of U-buffer (100 mm sodium Pi, pH 6.8, 10 mm Tris-HCl, 8 m urea, 0.1% Triton X-100). His6-ubiquitinated proteins were eluted with 50 μl of 2× Laemmli SB and 12–15 μl of samples were analyzed by Western blot with the relevant antibodies. In Vitro Ubiquitination Assay—A 150-ng aliquot of yeast E1 (Calbiochem), 150 ng of His6-Ubc4p, 200 ng of His6-Not4p, 1 μg of bovine ubiquitin (Fluka), and 1 μg of Egd1p or Egd2p or 2 μg of EDG complex were used in a 20-μl reaction containing 50 mm Tris-HCl, pH 7.5, 50 mm KCl, 2.5 mm MgCl2, 0.5 mm EDTA, 0.25 mm dithiothreitol, 2 mm ATP, 10 mm creatine phosphate, and 5 units of creatine phosphokinase (Calbiochem). In the first case, the EGD complex was co-expressed and co-purified, in the second case Egd1p and Egd2p were expressed and purified separately and then incubated for 1 h at 30 °C in an equimolar ratio, and added to the reaction. Reaction mixtures were incubated at 30 °C for 3 h and stopped with addition of 8 μl of 4× Laemmli SB. Live Cell Imaging—Transformed yeast cells were transferred on glass coverslips and analyzed on a Zeiss 510 confocal laser scanning microscope (LSM510). Images were recorded and image projections were assembled using the software package of the LSM 510 (Z-stack in all cases was 0.37 nm). The images were exported in tiff format and processed with Adobe Photoshop. The NAC and Ccr4-Not Complexes Interact—We first identified an interaction between the EGD and the Ccr4-Not complexes by the two-hybrid assay. We found that Egd1p could interact with all subunits of the Ccr4-Not complex in this assay (Fig. 1A). We used two different reporter systems, one for which the growth on plates lacking leucine is indicative of an interaction, but also one for which production of β-galactosidase is indicative of an interaction. Indeed, overproduction of certain subunits of the Ccr4-Not complex, such as Not2p, reduces cell growth, and this may prevent the visualization of a two-hybrid interaction that requires cell growth. Egd1p did not interact in this assay with several well expressed preys that consisted of subdomains of Ccr4-Not complex subunits, and the expression of the various subunits of the Ccr4-Not complex as preys did not in itself lead to transcriptional activation of the reporter genes (data not shown), indicating that the two-hybrid interactions measured were specific. The LexA-Egd2p fusion protein displayed low levels of activation and thus it was difficult to test for an interaction between the Ccr4-Not complex and Egd2p in this assay (data not shown). To analyze a possible interaction between the EGD and Ccr4-Not complexes further, we purified the EGD complex from total protein extracts of a strain that expressed Egd1p fused to 10 histidines from an episome to functionally complement the deletion of the endogenous EGD1 gene, using a nickel column. The column eluate was analyzed by Western blotting for the presence of both EGD subunits, of a ribosomal subunit L25, because Egd1p is known to be associated with ribosomes in vivo and with this subunit in particular (23Grallath S. Schwarz J.P. Bottcher U.M. Bracher A. Hartl F.U. Siegers K. EMBO Rep. 2006; 7: 78-84Crossref PubMed Scopus (21) Google Scholar), and for the presence of some of the subunits of the Ccr4-Not complex. We determined the presence of Egd1p and Egd2p as well as L25, Not4p, and Not5p, in the column eluate (Fig. 1B). We did not detect the presence of Not3p, another subunit of the Ccr4-Not complex that often tends to dissociate during purification of the Ccr4-Not complex (data not shown). None of these proteins were detected in the eluate of an extract prepared from the host cells without the plasmid. Thus, Not4p and Not5p from a total yeast extract can be retained on a column that binds the EGD complex. In addition we were able to co-precipitate Not1p and Not5p with Egd2p (data not shown). To determine whether the EGD complex could similarly be retained on a column that binds the Ccr4-Not complex, we created a strain expressing the Caf40p subunit of the Ccr4-Not complex fused with a GST entity. To purify the Ccr4-Not complex we prepared total protein extracts from cells expressing tagged Caf40p that we loaded on a GST-trap column. Bound proteins were eluted with reduced glutathione (Fig. 1C). The eluate of the affinity column was very dilute such that Egd2p, but not the subunits of the Ccr4-Not complex, were detected by Western blot, because of the quality of the antibodies against Egd2p. The eluate from the GST-trap column was then loaded on a MonoQ ion-exchange column, and eluted by a salt gradient. We determined the presence of all subunits of the Ccr4-Not complex, as well as Egd2p, in the same fraction of the MonoQ eluate (Caf40p, Not4p, and Egd2p are shown on Fig. 1C). Thus, Egd2p was co-purified through affinity and ion-exchange chromatography with the Ccr4-Not complex. Not4p Is Required for Ubiquitination of Egd1p and Egd2p—The experiments so far have demonstrated that the EGD complex interacts with the Ccr4-Not complex, which contains a potential E3 ligase subunit. We thus investigated whether Egd1p and Egd2p might be ubiquitinated proteins, and tested this in cells growing in high and low glucose, because the Ccr4-Not complex has been shown to play a role under both growth conditions. To undertake this study, we created a strain expressing Egd1p fused to a triple HA epitope from its own locus and promoter and transformed this strain with a plasmid expressing ubiquitin fused to 6 histidines. We then prepared total protein extracts under denaturing conditions from cells growing at different times with gradually decreasing levels of glucose concentration (from exponential phase (100 mm of glucose) to glucose depletion (no glucose)), and purified the total protein extracts on a nickel column. The eluate was analyzed by Western blot for the presence of Egd1p-HA3p with antibodies against the HA epitope. As a control, we similarly grew and tested the same strain prior to transformation (data not shown). As shown on Fig. 2A, we detected ubiquitinated forms of Egd1p-HA3p, particularly when glucose was decreased in the growth medium. This ubiquitination of Egd1p is not due to fusion of a tag to Egd1p, because using recently obtained antibodies against Egd1p, we saw similar ubiquitination of endogenous Egd1p after the diauxic shift in cells expressing endogenous Egd1p transformed with histidine-tagged ubiquitin (data not shown). To investigate a possible role of the Not4p E3 ligase in Egd1p ubiquitination, we disrupted the NOT4 gene in the strain expressing tagged Egd1p. In this strain, we integrated at the NOT4 locus a mutant allele of NOT4 expressing a RING finger mutant of Not4p that can no longer interact with the Ubc4p and Ubc5p E2 enzymes. 3K. W. Mulder, A. Inagaki, E. Cameroni, F. Mousson, G. S. Winkler, C. de Virgilio, M. A. Collart, and H. T. Timmers, submitted for publication. Both strains were transformed with the plasmid expressing histidine-tagged ubiquitin. Transformants were analyzed as before for Egd1p ubiquitination. We observed reduced levels of ubiquitinated Egd1p when NOT4 was disrupted, but no reduction when cells expressed a RING finger mutant of Not4p (Fig. 2A). The levels of Egd1p in the total extract were similar in the mutants and in the wild-type, and thus a decrease of total Egd1p cannot account for the reduced amount of ubiquitinated Egd1p observed in cells lacking Not4p. These results show that ubiquitination of Egd1p requires Not4p, but not its RING finger domain. The same cells were analyzed for Egd2p ubiquitination. In wild-type cells, we detected ubiquitinated forms of Egd2p, particularly when glucose was decreased in growth medium (Fig. 2B). Ubiquitinated forms of Egd2p were also detected in cells lacking Not4p, but to a greatly reduced extent (Fig. 2B). Furthermore, the extent of Egd2p ubiquitination was also somewhat reduced in cells expressing a RING finger mutant form of Not4p (Fig. 2B). The levels of Egd2p in the total extract were similar in mutants and wild-type, and thus a decrease of total Egd2p cannot account for the reduced amount of ubiquitinated Egd2p observed in the mutants. We next investigated the role of the other subunits of the Ccr4-Not complex in the ubiquitination of Egd2p, and for this we transformed each of the strains lacking one of the non-essential subunits of the Ccr4-Not complex with the plasmid expressing histidine-tagged ubiquitin. The transformants were tested as above. We were able to detect ubiquitinated forms of Egd2p in all strains, but to a lesser extent than in the wild-type cells, as shown in Fig. 2C for not2Δ, not3Δ, and not5Δ in addition to not4Δ. We additionally found that the level of ubiquitinated Egd2p was reduced in cells lacking Egd1p (Fig. 2C). As before, we confirmed that the levels of Egd2p in total extract were similar in all mutants and in the wild-type. Taken together these results indicate that expression of ubiquitinated Egd2p is dependent upon Not4p, its RING finger domain, the Ccr4-Not complex, and Egd1p. Not4p and Ubc4p Ubiquitinate the EGD Complex in Vitro—The results so far have shown that Not4p E3 ligase function is important for ubiquitination of the EGD complex in vivo. However, it remains possible that this effect is indirect, particularly because the RING finger domain of Not4p was not important for Egd1p ubiquitination in vivo (see above). To thus definitively determine whether Not4p can directly ubiquitinate the EGD complex, we wanted to determine whether Not4p could ubiquitinate the EGD complex in vitro. Because Not4p interacts with Ubc4p and Ubc5p enzymes, we first tested whether these E2 enzymes could interact with Egd1p and Egd2p in a two-hybrid assay. This was indeed the case (Fig. 3A). We were also able to co-immunoprecipitate Myc-tagged Ubc4p (and Ubc5p) with Egd2p, consistently in cells expressing Not4p, but less consistently in cells lacking Not4p (data not shown). These finding suggest that an interaction of Ubc4p (and Ubc5p) with Egd2p occurs in vivo, but is supported in large part by Not4p. Thus, we next purified recombinant Not4p, Ubc4p, Egd1p, Egd2p, and the EGD compl" @default.
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• W4234773207 title "The Yeast Ccr4-Not Complex Controls Ubiquitination of the Nascent-associated Polypeptide (NAC-EGD) Complex" @default.
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