Title: Physical and Functional Interaction between Elongator and the Chromatin-associated Kti12 Protein
Abstract: Cells lacking KTI12 or Elongator (ELP) genes are insensitive to the toxin zymocin and also share more general phenotypes. Moreover, data from low stringency immunoprecipitation experiments suggest that Elongator and Kti12 may interact. However, the precise relationship between these factors has not been determined. Here we use a variety of approaches to investigate the possibility that Elongator and Kti12 functionally overlap. Native Kti12 purified to virtual homogeneity under stringent conditions is a single polypeptide, but depletion of Kti12 from a yeast extract results in co-depletion of Elongator, indicating that these factors do interact. Indeed, biochemical evidence suggests that Elongator and Kti12 form a fragile complex under physiological salt conditions. Purified Kti12 does not affect Elongator histone acetyltransferase activity in vitro. However, a variety of genetic experiments comparing the effects of mutation in ELP3 and KTI12 alone and in combination with other transcription factor mutations clearly demonstrate a significant functional overlap between Elongator and Kti12 in vivo. Intriguingly, chromatin immunoprecipitation experiments show that Kti12 is associated with chromatin throughout the genome, even in non-transcribed regions and in the absence of Elongator. Conversely, RNA-immunoprecipitation experiments indicate that Kti12 only plays a minor role for Elongator association with active genes. Together, these experiments indicate a close physical and functional relationship between Elongator and the highly conserved Kti12 protein. Cells lacking KTI12 or Elongator (ELP) genes are insensitive to the toxin zymocin and also share more general phenotypes. Moreover, data from low stringency immunoprecipitation experiments suggest that Elongator and Kti12 may interact. However, the precise relationship between these factors has not been determined. Here we use a variety of approaches to investigate the possibility that Elongator and Kti12 functionally overlap. Native Kti12 purified to virtual homogeneity under stringent conditions is a single polypeptide, but depletion of Kti12 from a yeast extract results in co-depletion of Elongator, indicating that these factors do interact. Indeed, biochemical evidence suggests that Elongator and Kti12 form a fragile complex under physiological salt conditions. Purified Kti12 does not affect Elongator histone acetyltransferase activity in vitro. However, a variety of genetic experiments comparing the effects of mutation in ELP3 and KTI12 alone and in combination with other transcription factor mutations clearly demonstrate a significant functional overlap between Elongator and Kti12 in vivo. Intriguingly, chromatin immunoprecipitation experiments show that Kti12 is associated with chromatin throughout the genome, even in non-transcribed regions and in the absence of Elongator. Conversely, RNA-immunoprecipitation experiments indicate that Kti12 only plays a minor role for Elongator association with active genes. Together, these experiments indicate a close physical and functional relationship between Elongator and the highly conserved Kti12 protein. In competing for limited resources, microorganisms have evolved sophisticated strategies to gain selective advantage over their competitors. One of these is the secretion of toxic compounds that results in the killing or growth arrest of other species or genera. The yeast Kluyveromyces lactis secretes a toxin, referred to as zymocin, which inhibits the growth of various sensitive yeast genera, including Saccharomyces cerevisiae (1Stark M.J. Boyd A. Mileham A.J. Romanos M.A. Yeast. 1990; 6: 1-29Crossref PubMed Scopus (146) Google Scholar). The native toxin is a heterotrimeric (αβγ) structure composed of three subunits, two of which are involved in facilitating toxin entry. Cytotoxicity resides solely within the γ subunit, and intracellular expression of this subunit alone in S. cerevisiae abrogates growth (2Butler A.R. Porter M. Stark M.J. Yeast. 1991; 7: 617-625Crossref PubMed Scopus (52) Google Scholar). Genetic screening for mutations that confer resistance toward the intracellular expression of the zymocin γ subunit identified genes that were named TOT1–7 (toxin target) (3Jablonowski D. Frohloff F. Fichtner L. Stark M.J. Schaffrath R. Mol. Microbiol. 2001; 42: 1095-1105Crossref PubMed Scopus (62) Google Scholar, 4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar, 5Frohloff F. Fichtner L. Jablonowski D. Breunig K.D. Schaffrath R. EMBO J. 2001; 20: 1993-2003Crossref PubMed Scopus (136) Google Scholar). Interestingly, the initially isolated genes were found to encode subunits of either the yeast Elongator (Elp1/Tot1/Iki3, Elp2/Tot2, Elp3/Tot3, Elp4/Tot7, Elp5/Tot5/Iki1, and Elp6/Tot6) or the Kti12 protein (Tot4). ELP1/IKI3 and KTI12 were also isolated previously in independent screens for mutants that render cells resistant to the native toxin (Insensitive to Killer (IKI) and killer toxin-insensitive (KTI) genes, respectively) (2Butler A.R. Porter M. Stark M.J. Yeast. 1991; 7: 617-625Crossref PubMed Scopus (52) Google Scholar, 6Kishida M. Tokunaga M. Katayose Y. Yajima H. Kawamura-Watabe A. Hishinuma F. Biosci. Biotechnol. Biochem. 1996; 60: 798-801Crossref PubMed Scopus (54) Google Scholar). Elongator was first biochemically characterized as a component of the elongating form of RNA polymerase II (RNAPII) 1The abbreviations used are: RNAPII, RNA polymerase II; CTD, C-terminal domain; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; HA, hemagglutinin; HAT, histone acetyltransferase; RIP, RNA immunoprecipitation. 1The abbreviations used are: RNAPII, RNA polymerase II; CTD, C-terminal domain; ChIP, chromatin immunoprecipitation; GST, glutathione S-transferase; HA, hemagglutinin; HAT, histone acetyltransferase; RIP, RNA immunoprecipitation. (7Otero G. Fellows J. Li Y. de Bizemont T. Dirac A.M.G. Gustafsson C.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar) and contains the highly conserved histone acetyltransferase (HAT) Elp3 (8Wittschieben B.O. Otero G. de Bizemont T. Fellows J. Erdjument-Bromage H. Ohba R. Li Y. Allis C.D. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Recent evidence from both yeast and human cells indicates that Elongator interacts with active genes in vivo (9Gilbert C. Kristjuhan A. Winkler G.S. Svejstrup J.Q. Mol. Cell. 2004; 14: 457-464Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 10Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1247) Google Scholar). Interestingly, point mutations in Elp3, which abolish its HAT activity, also confer toxin resistance (5Frohloff F. Fichtner L. Jablonowski D. Breunig K.D. Schaffrath R. EMBO J. 2001; 20: 1993-2003Crossref PubMed Scopus (136) Google Scholar, 11Winkler G.S. Petrakis T.G. Ethelberg S. Tokunaga M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. J. Biol. Chem. 2001; 276: 32743-32749Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Moreover, mutagenesis studies on the ELP3 gene identified mutations outside the HAT domain, which confer sensitivity to killer toxin, but not the phenotypes otherwise typical of elp strains (separation of function mutations) (3Jablonowski D. Frohloff F. Fichtner L. Stark M.J. Schaffrath R. Mol. Microbiol. 2001; 42: 1095-1105Crossref PubMed Scopus (62) Google Scholar). This suggests that the requirement of Elongator for γ toxin sensitivity can be genetically dissociated from general Elongator function, perhaps through abolishing direct toxin-Elp3 interactions. The deletion of other HAT-encoding genes such as SAS3, HPA3, HAT1, and GCN5 does not confer zymocin resistance, further suggesting that Elp3, and not just any cellular HAT activity, is a target of the toxin (12Kitamoto H.K. Jablonowski D. Nagase J. Schaffrath R. Mol. Genet. Genomics. 2002; 268: 49-55Crossref PubMed Scopus (14) Google Scholar). In contrast to the deletion of ELP genes, which confers zymocin resistance, deletion of several genes encoding subunits of RNAPII transcription-related complexes renders cells toxin-hypersensitive (12Kitamoto H.K. Jablonowski D. Nagase J. Schaffrath R. Mol. Genet. Genomics. 2002; 268: 49-55Crossref PubMed Scopus (14) Google Scholar). This is true for genes that encode subunits of the SAGA, the SWI/SNF, the Mediator, and the Ccr4-Not complexes. Zymocin hypersensitivity is also observed in cells carrying deletions of transcript elongation-related factors such as ctk1 (RNAPII C-terminal domain (CTD) kinase 1), fcp1 (CTD phosphatase), and rtf1 (Paf complex) or mutations in rpb2 (RNAPII). In contrast, histone deacetylase-defective cells display either wild type or even reduced zymocin sensitivity. Based on the latter finding, Kitamoto et al. suggested that situations favoring histone hyperacetylation might reduce the cellular requirement for the HAT activity of Elongator and thereby reduce zymocin toxicity (12Kitamoto H.K. Jablonowski D. Nagase J. Schaffrath R. Mol. Genet. Genomics. 2002; 268: 49-55Crossref PubMed Scopus (14) Google Scholar). The above data suggest that the effect of the toxin is on RNAPII-dependent transcription. In apparent agreement with this idea, low resolution hybridization showed that global poly(A)+ mRNA levels generally decline in the presence of zymocin, and Northern blot analysis showed significantly reduced levels of specific RNAPII-generated transcripts (3Jablonowski D. Frohloff F. Fichtner L. Stark M.J. Schaffrath R. Mol. Microbiol. 2001; 42: 1095-1105Crossref PubMed Scopus (62) Google Scholar, 5Frohloff F. Fichtner L. Jablonowski D. Breunig K.D. Schaffrath R. EMBO J. 2001; 20: 1993-2003Crossref PubMed Scopus (136) Google Scholar). In an attempt to further reinforce the notion that zymocin action is linked to RNAPII, Jablonowski and Schaffrath studied the effects on zymocin toxicity of genetic conditions that would be supposed to directly impair polymerase activity, such as mutations in the RNAPII kinase encoded by the BUR1/BUR2 genes, deletion of the SRB10 CTD kinase gene, and inactivation of the kinase activity of TFIIH (13Jablonowski D. Schaffrath R. J. Biol. Chem. 2002; 277: 26276-26280Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In all cases, the mutant cells exhibited zymocin hypersensitivity. Moreover, hypersensitivity was also caused by truncation of the RNAPII CTD itself, further supporting the idea that a functional link between zymocin and RNAPII exists (13Jablonowski D. Schaffrath R. J. Biol. Chem. 2002; 277: 26276-26280Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). As mentioned above, KTI12 and the ELP genes were identified in a genetic screen using intracellular expression of the γ subunit as a means to look for targets of the toxin gene (TOT) (5Frohloff F. Fichtner L. Jablonowski D. Breunig K.D. Schaffrath R. EMBO J. 2001; 20: 1993-2003Crossref PubMed Scopus (136) Google Scholar). Cells lacking the KTI12/TOT4 gene display temperature sensitivity and 6-azauracil sensitivity as well as hypersensitivity to Calcofluor White and caffeine (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar). Thus, kti12 cells have phenotypes that are similar to those observed for elp cells (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar, 7Otero G. Fellows J. Li Y. de Bizemont T. Dirac A.M.G. Gustafsson C.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Low stringency co-immunoprecipitation experiments suggested the existence of an interaction between Kti12 and Elongator as well as one between Kti12 and the form of RNAP II phosphorylated at serine 5 of the CTD repeat (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar, 14Frohloff F. Jablonowski D. Fichtner L. Schaffrath R. J. Biol. Chem. 2003; 278: 956-961Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, deletion of KTI12 does not appear to affect the structural integrity of six-subunit Elongator complex (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar), making it unlikely that Kti12 is a structural component of Elongator. Interestingly, chromatin immunoprecipitation (ChIP) experiments suggested that Kti12 occupies the promoter, but not the coding region, of the ADH1 gene (15Fichtner L. Frohloff F. Jablonowski D. Stark M.J. Schaffrath R. Mol. Microbiol. 2002; 45: 817-826Crossref PubMed Scopus (36) Google Scholar). We were intrigued by the data suggesting a functional connection between Kti12 and Elongator. Our previous data showed no evidence for Kti12 in highly purified Elongator fractions (11Winkler G.S. Petrakis T.G. Ethelberg S. Tokunaga M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. J. Biol. Chem. 2001; 276: 32743-32749Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and the apparent overlap of elp and kti12 phenotypes might conceivably be misleading. Moreover, the immunoprecipitation experiments suggesting a Kti12-Elongator interaction were performed under non-stringent conditions that permit detection also of less meaningful protein-protein interactions. In this paper we set out to investigate in more detail the possibility that Elongator and Kti12 functionally overlap. Our data are consistent with the idea that Elongator and Kti12 interact in a manner that has important consequences for the function of Elongator as a histone acetyltransferase in vivo. Our data also suggest that Kti12 is a general chromatin component rather than a promoter-specific or even a gene-specific factor as proposed previously (15Fichtner L. Frohloff F. Jablonowski D. Stark M.J. Schaffrath R. Mol. Microbiol. 2002; 45: 817-826Crossref PubMed Scopus (36) Google Scholar). Yeast Strains and Phenotypic Analysis—All S. cerevisiae stains used for genetic analysis were congenic with strain W303 and grown and manipulated as described previously (7Otero G. Fellows J. Li Y. de Bizemont T. Dirac A.M.G. Gustafsson C.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 16Wittschieben B.O. Fellows J. Du W. Stillman D.J. Svejstrup J.Q. EMBO J. 2000; 19: 3060-3068Crossref PubMed Scopus (122) Google Scholar). Genotypes of the strains used are shown in Table I.Table IGenotypes of the strains used in this studyS. cerevisiae strainsGenotypeSourceJSY130MATα elp3Δ::LEU2(8Wittschieben B.O. Otero G. de Bizemont T. Fellows J. Erdjument-Bromage H. Ohba R. Li Y. Allis C.D. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar)JSY959MATα kti12Δ::URA3This studyJSY1021MATα kti12Δ::URA3 elp3Δ::LEU2This studyJSY142MATα gcn5Δ::HIS3(16Wittschieben B.O. Fellows J. Du W. Stillman D.J. Svejstrup J.Q. EMBO J. 2000; 19: 3060-3068Crossref PubMed Scopus (122) Google Scholar)JSY144MATα elp3Δ::LEU2 gcn5Δ::HIS3(16Wittschieben B.O. Fellows J. Du W. Stillman D.J. Svejstrup J.Q. EMBO J. 2000; 19: 3060-3068Crossref PubMed Scopus (122) Google Scholar)JSY970MATa kti12Δ::URA3This studyElp1(MYC18)::HIS3JSY960MATα KTI12(HA6)::HIS3This studyJSY993MATα KTI12(MYC18)::URA3This studyJSY996MATα KTI12(His10-HA)::TRP1This studyJSY994MATα elp2Δ::LEU2This studyKTI12(MYC18)::URA3JSY995MATα elp3Δ::LEU2This studyKTI12(MYC18)::URA3JSY1001MATα kti12Δ::URA3 gcn5Δ::HIS3This studyJSY991MATα kti12Δ::URA3 gcn5Δ::HIS3This studyelp3Δ::LEU2JSY992MATa kti12Δ::URA3 gcn5Δ::HIS3This studyhos2Δ::TRP1 hda1Δ::KANMXK. lactis strainsIFO 1267aKind gift from Prof. Michael J. R. StarkMATa (toxic)MBK 801aKind gift from Prof. Michael J. R. StarkMATa (non-toxic)a Kind gift from Prof. Michael J. R. Stark Open table in a new tab Expression of Tagged Proteins in Vivo—For the construction of the Kti12-HisHA strain, part of the KTI12 open reading frame was amplified by PCR and cloned into pSE.HISHA-304 (17Winkler S.G. Lacomis L. Philip J. Erdjument-Bromage H. Svejstrup J.Q. Tempst P. Methods. 2002; 26: 260-269Crossref PubMed Scopus (70) Google Scholar) using the KpnI and BamHI sites to produce plasmid pKTI12-HISHA-304. After yeast transformation, a TRP+ clone was isolated in which the 3′-end of the KTI12 gene was replaced, resulting in expression of a Kti12-(His)10-HA fusion protein. Phenotypic analysis showed that the (His)10-HA epitope tag did not interfere with Kti12 function (data not shown). Similar procedures, but using tagging plasmids kindly supplied by Dr. Kim Nasmyth (18Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (812) Google Scholar), were used to produce and characterize Kti12–6HA and Kti12–18Myc strains (oligonucleotide sequences and other details are available on request). Protein Purification—The procedure for purification of Kti12 from a KTI12-HISHA strain has been described elsewhere (11Winkler G.S. Petrakis T.G. Ethelberg S. Tokunaga M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. J. Biol. Chem. 2001; 276: 32743-32749Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Winkler S.G. Lacomis L. Philip J. Erdjument-Bromage H. Svejstrup J.Q. Tempst P. Methods. 2002; 26: 260-269Crossref PubMed Scopus (70) Google Scholar). Gel filtration analysis was performed using a Superose 6 column (Amersham Biosciences) connected to a Biologic fast protein liquid chromatography system (Bio-Rad). The buffer used was 2% glycerol, 250 mm potassium acetate, pH 7.6, 0.1% Nonidet P-40, and 1× protease inhibitors. The column was run at a flow rate of 30 μl/min, and the protein-containing fractions were analyzed by Western blotting. Size markers (Amersham Biosciences) were dissolved in the same buffer and run immediately before and after each experimental sample for reference. Protein Identification—Gel-fractionated proteins were digested with trypsin and peptides analyzed by matrix-assisted laser-desorption/ionization reflectron time-of-flight mass spectrometry (MALDI-TOF) and by electrospray ionization tandem mass spectroscopy as previously described (17Winkler S.G. Lacomis L. Philip J. Erdjument-Bromage H. Svejstrup J.Q. Tempst P. Methods. 2002; 26: 260-269Crossref PubMed Scopus (70) Google Scholar). Selected mass values from the MALDI-TOF experiments were taken to search the protein non-redundant data base (National Center for Biotechnology Information, Bethesda, MD) using the PeptideSearch (19Mann M. Hojrup P. Roepstorff P. Biol. Mass Spectrom. 1993; 22: 338-345Crossref PubMed Scopus (833) Google Scholar) algorithm. Tandem mass spectrometry spectra were inspected for y″ ion series to compare with the computer-generated fragment ion series of the predicted tryptic peptides. Co-immunoprecipitation Experiments—500 μg of yeast whole cell extract in buffer A (40 mm Hepes-KOH, pH 7.6, 1 mm EDTA, 1 mm dithiothreitol, 20% (v/v) glycerol, and protein inhibitor mix) containing 250 or 500 mm potassium acetate, as indicated, was incubated for 2 h with Sepharose A beads, which had been previously conjugated with the 12CA5 antibody. After incubation, the beads were washed three times with the same buffer, re-suspended in 1× SDS loading buffer, and the bound proteins were subjected to SDS-PAGE and Western blot analysis. Expression of GST-Kti12 in Bacteria and Antibody Production—The Kti12 open reading frame was cloned in-frame with the GST protein in pGEX-3X and the fusion protein expressed in Escherichia coli BL21 DE3 cells by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 6 h at 28 °C. Subsequently, cells were lysed in phosphate-buffered saline, and inclusion bodies were solubilized by sonication of the pellet in the presence of 0.5% sarcosyl. Solubilized GST-Kti12 was purified on glutathione-Sepharose per the manufacturer's instructions (Amersham Biosciences). The recombinant fusion protein was used to immunize rabbits (Imgenex). The resulting antibody was used for Western blots at 1:1000 final dilution in phosphate-buffered saline containing 0.05% Tween and 5% (w/v) milk powder. Killer Toxin Assays—To analyze killer toxin sensitivity, K. lactis cells expressing or not expressing the zymocin toxin were left to grow overnight on YPD (1% yeast extract, 2% peptone, and 2% dextrose) medium (strains used are shown in Table I). The next day, ∼10,000 S. cerevisiae cells were dissolved in water and spotted in the vicinity of the growing K. lactis cells. The growth of the different mutants was compared with the characteristic eclipse growth of wild type S. cerevisiae cells. Other Assays—Histone acetyltransferase reactions (30 μl) were carried out as described (11Winkler G.S. Petrakis T.G. Ethelberg S. Tokunaga M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. J. Biol. Chem. 2001; 276: 32743-32749Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Chromatin and RNA immunoprecipitation experiments were performed as described (9Gilbert C. Kristjuhan A. Winkler G.S. Svejstrup J.Q. Mol. Cell. 2004; 14: 457-464Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Petrakis T.G. Wittschieben B.O. Svejstrup J.Q. J. Biol. Chem. 2004; 279: 32087-32092Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 21Kristjuhan A. Walker J. Suka N. Grunstein M. Roberts D. Cairns B.R. Svejstrup J.Q. Mol. Cell. 2002; 10: 925-933Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Oligonucleotide primer sequences are available on request. Purification of Native Yeast Kti12—Previous work by Schaffrath and co-workers reported evidence suggesting an Elongator-Kti12 interaction (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar). However, whether a small subset of Elongator complexes contained Kti12 as an integral, tightly associated subunit or whether Kti12 was merely weakly interacting with Elongator remained unclear. Moreover, the experiments suggesting the interaction were performed under very low stringency conditions (60 mm sodium acetate) (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar), which might result in the detection of interactions that are not biologically significant. We tested the possibility that Kti12 and Elongator might exist in the same complex by isolating native yeast Kti12. The genomic KTI12 gene was modified so that the expressed Kti12 protein carried a C-terminal (His)10-HA tag. After genetic confirmation that the epitope tag did not interfere with Kti12 function (data not shown), the protein was purified by a mixture of conventional and affinity chromatography (Fig. 1A), as described previously (11Winkler G.S. Petrakis T.G. Ethelberg S. Tokunaga M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. J. Biol. Chem. 2001; 276: 32743-32749Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 17Winkler S.G. Lacomis L. Philip J. Erdjument-Bromage H. Svejstrup J.Q. Tempst P. Methods. 2002; 26: 260-269Crossref PubMed Scopus (70) Google Scholar). Fig. 1B shows a Coomassie-stained SDS-polyacrylamide gel of highly purified Kti12. The two polypeptides, of 40 and 180 kDa, respectively, were identified by mass spectrometry. As expected, the 40-kDa protein was Kti12. The 180-kDa polypeptide was identified as the product of the YDL223C gene. This protein is a frequent, irrelevant contaminant when this purification procedure is utilized (22Gilbert C.S. van den Bosch M. Green C.M. Vialard J.E. Grenon M. Erdjument-Bromage H. Tempst P. Lowndes N.F. EMBO Rep. 2003; 4: 953-958Crossref PubMed Scopus (21) Google Scholar, 23Fellows J. Erdjument-Bromage H. Tempst P. Svejstrup J. J. Biol. Chem. 2000; 275: 12896-12899Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A rabbit anti-Kti12 antibody, but not the corresponding rabbit pre-bleed, recognized the highly purified Kti12-HisHA protein isolated from yeast as well as the recombinant, bacterially expressed GST-Kti12 protein (Fig. 1C, anti-Kti12, lanes 2 and 3, respectively). Using this antibody, we also found that the highly purified holo-Elongator complex did not contain Kti12 (Fig. 1C, anti-Ktil2, lane 1). Conversely, the highly purified yeast Kti12 did not contain Elongator, as indicated by the absence of Elp3 and Elp4 (Fig. 1D, lane 2). These data indicate that Kti12 is not a tightly associated, integral subunit of holo-Elongator complexes in general. Moreover, the finding that purification of Kti12 to virtual homogeneity did not uncover co-purifying proteins indicates that this protein is also not a stable component of other protein complexes. Elongator and Kti12 Interact in a Salt-labile Manner—The method used to purify holo-Elongator and Kti12 made it impossible to exclude the possibility that a weak, salt-labile interaction exists between these proteins. To investigate possible Kti12-Elongator interactions, co-immunoprecipitation experiments were performed using extracts from cells expressing a version of the Kti12 protein that carried a 6× HA affinity tag. Fig. 2 shows the Western blot analysis of immunoprecipitations with 12CA5 (anti-HA) antibody using extracts from cells expressing untagged and tagged Kti12, respectively. In the control immunoprecipitation from untagged cells, none of the Elongator proteins were detected in the precipitates (Fig. 2A, lane 3), whereas 6× HA-tagged Kti12 effectively co-immunoprecipitated Elp3 and Elp4 under low salt conditions (Fig. 2, lane 6, 250 mm potassium acetate). Under more stringent conditions (Fig. 2, lane 8, 500 mm potassium acetate), there was significantly less Elongator associated with Kti12. However, both Elp3 and Kti12 were clearly co-depleted from the resin flow-through under both conditions (Fig. 2A, compare lanes 5 and 7 with lane 4), suggesting that Elongator and Kti12 do indeed interact but dissociate during the salt wash. Based on these observations, we now sought to purify the Kti12-HisHA protein under less stringent conditions (250 mm salt) (Fig. 3). In particular, as the majority of Kti12 elutes from Bio-Rex in 600 mm salt, this fraction was diluted to 250 mm prior to loading on 12CA5-conjugated Sepharose A beads (see Fig. 1A). After washing this column with buffer containing 250 mm potassium acetate, proteins were eluted and subjected immediately to nickel-agarose affinity chromatography. The resulting nickel-agarose elution profile was examined by Western blot analysis. Strikingly, Kti12 and Elp3 were now found to co-elute from the final nickel-agarose purification step (Fig. 3A). Unfortunately, because of the lower stringency of the purification procedure, other proteins contaminated the fraction; thus, to investigate whether Elp3 and Kti12 were in the same complex, the proteins in the 500 mm imidazole elution fraction were subjected to gel filtration chromatography. Fig. 3B shows a Western blot analysis of the sizing column elution profile. Although the resolution of this particular gel filtration experiment was not very good, several observations could be made. First, there was a good, but not precise, overlap of the elution profiles for Elp3 and Kti12 (Elongator peaks in fraction 16, whereas Kti12 peaks in fraction 18, Fig. 3B). Second, Kti12 eluted as a protein of much higher molecular mass than expected from its predicted size (37 kDa). The fraction in which Elongator peaked (Fig. 3B, fraction 18) also contained significant amounts of Kti12, consistent with the idea that under low stringency conditions Kti12 may associate with Elongator. Finally, the elution of "Elongator-free" Kti12 at molecular masses significantly above 37 kDa suggests that Kti12 either multimerizes or co-elutes with other proteins under those conditions. However, an alternative (and more likely) explanation is that a weak complex between Elongator and Kti12 is dissociating as a consequence of the dilution occurring during the course of the gel filtration experiment, resulting in their slight separation. Taken together, these data indicate that Kti12 associates with Elongator in a salt-labile manner. Genetic Interactions between Kti12 and Gcn5—The above results indicate that Elongator and Kti12 interact physically. Previous genetic characterization has shown that strains lacking KTI12 share number of general phenotypes with strains lacking ELP genes (4Fichtner L. Frohloff F. Burkner K. Larsen M. Breunig K.D. Schaffrath R. Mol. Microbiol. 2002; 43: 783-791Crossref PubMed Scopus (60) Google Scholar). We set out to more precisely define the functional overlap between Elongator and Kti12 in vivo. We hypothesized that if Kti12 plays a role in the same cellular pathway as Elongator, then deletion of KTI12 should not result in any further deterioration in the growth of an elp3 strain. Indeed, cells lacking both ELP3 and KTI12 (elp3 kti12) displayed growth rates that were no worse than the kti12 and elp3 single mutants under a number of different conditions (Fig. 4A, and data not shown). We also surmised that if Kti12 is an important functional partner of Elongator in the cell, it should genetically interact with Gcn5, as Elongator does (16Wittschie