Characterization of CAF4 and CAF16 Reveals a Functional Connection between the CCR4-NOT Complex and a Subset of SRB Proteins of the RNA Polymerase II Holoenzyme
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m009112200
ISSN1083-351X
AutoresHaiyan Liu, Y. C. Chiang, Jing Pan, Junji Chen, Christopher Salvadore, Deborah C. Audino, Vasudeo Badarinarayana, Palaniswamy Viswanathan, Bradley D. Anderson, Clyde L. Denis,
Tópico(s)RNA modifications and cancer
ResumoThe CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 × 106dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex. We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex. CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex. In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduceADH2 derepression and either an srb9 orsrb10 allele suppressed spt10-enhanced expression of ADH2. In addition, srb9 andsrb10 alleles increasedADR1 c-dependent ADH2expression; not4 and not5 deletions are the only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme. The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 × 106dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex. We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex. CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex. In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduceADH2 derepression and either an srb9 orsrb10 allele suppressed spt10-enhanced expression of ADH2. In addition, srb9 andsrb10 alleles increasedADR1 c-dependent ADH2expression; not4 and not5 deletions are the only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme. TATA box protein The CCR4-NOT complex is one of several large groups of proteins involved in transcription (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar). It consists of at least two complexes, 1.9 and 1.0 MDa in size, that are distinct from other large, transcriptionally important groups of proteins, such as the SNF/SWI complex, the SAGA complex, TFIID, and the RNA polymerase II holoenzyme (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 2Denis C.L. Draper M.P. Liu H.-Y. Malvar T. Vallari R.C. Cook W.J. Genetics. 1994; 138: 1005-1013Crossref PubMed Google Scholar, 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar). 1H.-Y. Liu, Y.-C. Chiang, J. Chen, V. Badarinarayana, and C. L. Denis, unpublished observations. The smaller of the CCR4-NOT complexes contains CCR4, CAF1 (POP2), the five NOT proteins (NOT1–5), and two other proteins (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar).1These proteins can all be coimmunoprecipitated with antibody specific to either CCR4, CAF1, or NOT proteins (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar). All of the components of the 1-MDa CCR4-NOT complex also comigrate at 1.9 MDa following gel filtration analysis (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar),1 and mutations in individual components of the CCR4-NOT complex destroy the ability of these components to migrate at 1.9 MDa (4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar).1 Although antibody directed against the core CCR4-NOT proteins does not coimmunoprecipitate any other proteins, a number of other proteins that do not coimmunoprecipitate at their physiological concentrations with CCR4 antibody do interact with the CCR4-NOT complex genetically and can be coimmunoprecipitated when overexpressed. These proteins may possibly be components of the larger 1.9-MDa complex and include such proteins as DBF2, a cell cycle-regulated protein kinase (5Liu H.-Y Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (88) Google Scholar), MOB1, a protein that binds DBF2 and is involved in cell cycle regulation (6Komarnitsky S.I. Chiang Y.-C. Luca F.C. Chen J. Toyn J.H. Winey M. Johnston L.H. Denis C.L. Mol. Cell. Biol. 1998; 18: 2100-2107Crossref PubMed Scopus (88) Google Scholar), and DHH1, a putative RNA helicase (7Hata H. Mitsui H. Liu H.-Y. Bai Y. Denis C.L. Shimizu Y. Sakai A. Genetics. 1998; 148: 571-579PubMed Google Scholar). Both DBF2 and MOB1 have also been observed to migrate at 1.9 MDa following gel filtration analysis.1 The internal arrangement of factors in the 1-MDa CCR4-NOT complex has been studied (4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar). NOT1 appears to be the core component of the complex. CAF1 binds to the central region of NOT1 and links CCR4 to the rest of the NOT proteins. The C terminus of NOT1, in turn, contacts NOT2, NOT5, and NOT4. The physical separation of CCR4 and CAF1 from NOT2, NOT4, and NOT5 agrees with several phenotypic differences between these proteins (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar). Whereas CAF1 is absolutely required for CCR4 to associate with the 1-MDa complex (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar), CCR4 can still associate in the 1.9-MDa complex in the absence of CAF1 (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar). It is likely, therefore, that within this larger complex CCR4 is making contacts to proteins other than ones found in the core 1-MDa complex. Based on the observation that CCR4 can only immunoprecipitate other components of the core 1-MDa complex, these other interactions in the 1.9-MDa complex may be more susceptible to disruption. The CCR4-NOT proteins have been found to affect gene expression both positively and negatively (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 8Collart M.A. Mol. Cell. Biol. 1996; 16: 6668-6676Crossref PubMed Scopus (101) Google Scholar, 9Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (173) Google Scholar, 10Denis C.L. Genetics. 1984; 108: 833-844Crossref PubMed Google Scholar, 11Oberholzer U. Collart M.A. Gene ( Amst. ). 1998; 207: 61-69Crossref PubMed Scopus (57) Google Scholar, 12Sakai A. Chibazakura T. Shimizu Y. Hishinuma F. Nucleic Acids Res. 1992; 20: 6227-6233Crossref PubMed Scopus (58) Google Scholar, 13Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 487-495Crossref Scopus (96) Google Scholar). Their action as repressors are likely to be the result of the NOT proteins restricting access of TBP2 to noncanonical TATAAs (8Collart M.A. Mol. Cell. Biol. 1996; 16: 6668-6676Crossref PubMed Scopus (101) Google Scholar, 9Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (173) Google Scholar). NOT1 has been shown to associate with TBP (14Lee T.I. Wyric J.J. Koh S.S. Jennings E. Gadbois E.L. Young R.A. Mol. Cell. Biol. 1998; 18: 4455-4462Crossref PubMed Scopus (85) Google Scholar); NOT5 interacts with TFIID (15Badarinarayana V. Chiang Y.-C. Denis C.L. Genetics. 2000; 155: 1045-1054Crossref PubMed Google Scholar, 47Schneider B.L. Seufert W. Steiner B. Yang Q.H. Futcher A.B. Yeast. 1995; 11: 1265-1274Crossref PubMed Scopus (291) Google Scholar), and NOT2 has been shown to associate with ADA2, a component of the SAGA complex (16Benson J.D. Benson M. Howley P.M. Struhl K. EMBO J. 1998; 17: 6714-6722Crossref PubMed Scopus (43) Google Scholar). Consistent with these results, deletions of upstream sequences do not apparently affect the ability of CCR4 to affect ADH2 expression (17Denis C.L. Malvar T. Genetics. 1990; 124: 283-291Crossref PubMed Google Scholar), and it has been shown that CCR4 acts at a post-chromatin remodeling step in affectingADH2 derepression (18Verdone L. Cesari F. Denis C.L. Di. Mauro E. Caserta M. J. Biol. Chem. 1997; 272: 30828-30834Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The large sizes of the CCR4-NOT complexes and the possible mode of action of these proteins at or near the TATAA suggest that these complexes would be likely to interact with other proteins acting to control initiation of transcription. In this study we report the identification of two additional factors, CAF4 and CAF16, that interact with the CCR4-NOT proteins and that affect transcription both positively and negatively. Neither CAF16 nor CAF4 was a component of the 1-MDa CCR4-NOT complex, but both proteins were present in a 1.9-MDa complex, and their presence in this complex was dependent on CCR4. CAF16 and CAF4, in turn, were found to interact with SRB9, -10, and -11, components of the RNA polymerase II holoenzyme (19Balciunas D. Ronne H. Nucleic Acids Res. 1995; 23: 4421-4425Crossref PubMed Scopus (60) Google Scholar, 20Carlson M. Annu. Rev. Cell. Dev. Biol. 1997; 13: 1-23Crossref PubMed Scopus (179) Google Scholar, 21Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.-M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar, 22Song W. Treich I. Qian N. Kuchin S. Carlson M. Mol. Cell. Biol. 1996; 16: 115-120Crossref PubMed Scopus (110) Google Scholar, 23Sun X. Zhang Y. Cho H. Rickert P. Lees E. Lane W. Reinberg D. Mol. Cell. 1998; 2: 213-222Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). SRB10 and -11 are a cyclin-dependent protein kinase/cyclin pair that are capable of phosphorylating the CTD of RNA polymerase II (21Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.-M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar, 24Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 1163-1171Crossref PubMed Scopus (111) Google Scholar,25Liao S.-M. Zhang J. Jeffery D.A. Koleske A.J. Thompson C.M. Chao D.M. Viljoen M. Van. Vuuren H.J.J. Young R.A. Nature. 1995; 374: 193-196Crossref PubMed Scopus (367) Google Scholar). Whereas none of these three SRB genes are essential, the SRB9–11 proteins have been found to play both positive and negative roles in transcription (20Carlson M. Annu. Rev. Cell. Dev. Biol. 1997; 13: 1-23Crossref PubMed Scopus (179) Google Scholar, 26Berk A.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11952-11954Crossref PubMed Scopus (7) Google Scholar, 27Cooper K.F. Mallory M.J. Smith J.B. Strich R. EMBO J. 1997; 16: 4665-4675Crossref PubMed Scopus (97) Google Scholar, 28Wahi M. Johnson A.D. Genetics. 1995; 140: 79-90Crossref PubMed Google Scholar), although they appear to be more predominantly involved in repressing transcription (21Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.-M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar, 22Song W. Treich I. Qian N. Kuchin S. Carlson M. Mol. Cell. Biol. 1996; 16: 115-120Crossref PubMed Scopus (110) Google Scholar, 23Sun X. Zhang Y. Cho H. Rickert P. Lees E. Lane W. Reinberg D. Mol. Cell. 1998; 2: 213-222Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). We found that the SRB9, 10, and 11 proteins can coimmunoprecipitate the CCR4 and NOT proteins, and defects in these SRB proteins affect expression at the ADH2 locus in a manner similar to that observed for defects in CCR4 complex components. These results indicate a close physical and functional link between CCR4-NOT components and the SRB9, -10, and -11 proteins. Yeast strains are listed in TableI.Table IYeast strainsStrainGenotypeCHY109Matα srb9::URA3 ura3–52 his3–200 leu2–3,112 rpb1-187::HIS3 [pRB114-RPB-LEU2]EGY188MATa ura3 his3 trp1 LexAopLEU2EGY188–1Isogenic to EGY188 exceptccr4::URA3EGY188-c1Isogenic to EGY188 except caf1::URA3EGY188-c4Isogenic to EGY188 except caf4::URA3EGY188–16aIsogenic to EGY188 exceptcaf16::URA3EGY191MATα ura3 his3 trp1 LexAopLEU21166-2-6aMATa adh1-11 ura3 trp1 his3 srb9::URA3994-2MATα adh1-11 ura3 trp1 his3 leu2 spt10::TRP11357-4dMATα adh1-11 ura3 trp1 leu2 his3 srb10::HIS31366-3dMATa adh1::URA3 ura3 trp1 leu2 his3 spt10::LEU21188-1aMATa adh1-11 ura3 trp1 his3 spt10::TRP11188-1bMATα adh1-11 ura3 trp1 leu2 srb9::URA3787-11aMATa adh1-11 ADR1-5c ura3 trp1 leu2 his31240-2-7cMATa adh1-11 ura3 his3 leu2 trp1 srb9::URA3612–1dMATa adh1–11 ura3 his3 leu2 trp1787–6bMATα adh1–11 ura3 his3 leu2 trp1 ADR1–5c-TRP11637–2bMATa ura3 his3 leu2 trp1 dbf2::DBF2–6c-myc-URA31637–2b-1aIsogenic to 1637–2b exceptccr4::HIS31588–2dMATα ura3 his3 leu2 trp1 caf4::CAF4-HA-URA31588–2d-1aIsogenic to 1588–2d except ccr4::HIS3 Open table in a new tab CAF16 was sequenced on each strand by double-stranded sequencing using Sequenase (U. S. Biochemical Corp.). Sequence comparison analysis was performed at the National Center for Biotechnology Information, using the BLAST network service MegAlign version 1.05 (BLAST version, 1.8.1). Alignments were performed by using the Clustal method available in the DNASTAR package (DNA Star Inc.). A Superose 6 column HR10/30 was used according to the manufacturer's instructions (Amersham Pharmacia Biotech). Two hundred μl of yeast extract at a concentration of about 10 mg/ml was placed over the column following clarification by centrifugation as described previously (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar). Running conditions were as described (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar), and the standards were eluted as follows: blue dextran (2000 kDa) at 10 ml; thyroglobulin (669 kDa) at 15 ml; bovine serum albumin (66 kDa) at 17.5 ml. Immunoprecipitations were carried out as described previously (3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar, 5Liu H.-Y Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (88) Google Scholar). Western analyses were conducted as described (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar). Antibody to CAF16 was raised against its C-terminal peptide, CCKRDNQIPDKEIGI, whereas antibody to CAF4 was against CAF4 tagged with three copies of the HA1 epitope at its C terminus and integrated at the CAF4 locus as described (46Hengartner C.J. Myer V.E. Liao S.-M. Wilson C.J. Koh S.S. Young R.A. Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). Thecaf4::URA3 disruption plasmid was created by replacing the BclI fragment of CAF4 (base pairs 851–2162) with a BamHI fragment of URA3. TheCAF16 disruption plasmid was constructed by removing nucleotides 368–706 base pairs of CAF16 following cutting with NdeI, blunt ending with the large subunit ofEscherichia coli DNA polymerase (Klenow), and replacing it with a HindIII fragment of URA3 (blunt-ended with Klenow). The resultant plasmid JP7 was cut with M1uI andSstI prior to transformation to replace the chromosomal copy of CAF16. LexA-CAF16 (pJp1) was constructed by placing an EcoRI fragment, 2 kilobase pairs, from pRS316-CAF16 into the EcoRI polylinker of pLexA87- (9Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (173) Google Scholar). LexA-CAF4 was formed by ligating theEcoRI fragment of CAF4 from ML4-3 (2 kilobase pairs EcoRV piece of CAF4 cloned into theSmaI site of pSP72) into LexA-202-2 (9Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (173) Google Scholar) cut withEcoRI. ADH and β-galactosidase activities were determined as previously described (29Cook W.J. Chase D. Audino D.C. Denis C.L. Mol. Cell. Biol. 1994; 14: 629-640Crossref PubMed Google Scholar). The screen for proteins interacting with LexA-CCR4 (full-length) was conducted as described previously using strain EGY188 containing the p34 reporter plasmid (8 LexA operators upstream of thelacZ gene). The B42 fusion proteins contain the HA1 epitope, the B42 E. coli-derived activation domain, a nuclear localization sequence, and fragments of the yeast genome fused to the C terminus of B42 (30Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 232-233Abstract Full Text PDF Scopus (666) Google Scholar). The LexA activators were LexA-ADR1 containing full-length ADR1, LexA-ADR1-TADIV containing residues 642–704 of ADR1, LexA-B42 containing the E. coli-derived B42 activation domain fused to LexA-(1–202) (29Cook W.J. Chase D. Audino D.C. Denis C.L. Mol. Cell. Biol. 1994; 14: 629-640Crossref PubMed Google Scholar), LexA-SRB9, -10, and -11 (24Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 1163-1171Crossref PubMed Scopus (111) Google Scholar) containing full-length versions of the respective SRB proteins, and LexA-CCR4-(1–344) containing residues 1–344 of CCR4 (5Liu H.-Y Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (88) Google Scholar). A yeast two-hybrid screen using LexA-CCR4 as a bait was conducted with a library of yeast sequences fused to the B42 activator to identify additional components of the multisubunit CCR4-NOT complexes. In addition to identifying CAF1 and DBF2 (5Liu H.-Y Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (88) Google Scholar, 13Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 487-495Crossref Scopus (96) Google Scholar) as components or associated factors of the CCR4-NOT complex, two additional proteins (designated CAF4 and CAF16) were found to interact with LexA-CCR4 and not with LexA alone (Table II, lines 2 and 3 compared with line 1). Deletion analysis indicated that an intact leucine-rich repeat (residues 345–470) of CCR4 was required for its interaction with both CAF4 and CAF16 (Table II, lines 7 and 8) but not the N-terminal region of CCR4 (lines 5 and 6); the leucine-rich repeat was also required for CCR4 interaction with CAF1, NOT1, and DBF2 (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar,5Liu H.-Y Toyn J.H. Chiang Y.-C. Draper M.P. Johnston L.H. Denis C.L. EMBO J. 1997; 16: 5289-5298Crossref PubMed Scopus (88) Google Scholar, 13Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 487-495Crossref Scopus (96) Google Scholar). Sequencing of CAF4 revealed it to encode a novel protein (yeast protein YKR036c) containing seven WD40 repeats in its C terminus (residues 320–659). CAF16 when sequenced in its entirety was found to encode a protein (now designated YFL028c) that shares significant homology to the ABC ATPase family of proteins (31Dean M. Allikmets R. Gerrard B. Stewart C. Kistler A. Sharer B. Michaelis S. Strathern J. Yeast. 1994; 10: 377-383Crossref PubMed Scopus (58) Google Scholar,32Poole R.K. Hatch L. Cleeter M.W. Gibson F. Cox G.B. Wu G. Mol. Microbiol. 1993; 10: 421-430Crossref Scopus (74) Google Scholar). ABC ATPases are principally found to play roles in transport across membranes and as membrane receptors (33Decottignies A. Goffeau A. Nat. Genet. 1997; 15: 137-145Crossref PubMed Scopus (394) Google Scholar). CAF16 differs from most of the eucaryotic ABC ATPase proteins in that it lacks the transmembrane domains characteristic of this family. One other eucaryotic ABC ATPase, EF3, involved in translational elongation also lacks these signature transmembrane domains (34Qin S. Xie A. Bonato C.M. McLaughlin C.S. J. Biol. Chem. 1990; 265: 1903-1912Abstract Full Text PDF PubMed Google Scholar). CAF16 also contains only one ABC ATPase domain, whereas most other eucaryotic ABC ATPases contain two domains, suggesting that CAF16 may interact with itself, as was confirmed by two-hybrid analysis (Table II, line 14).Table IICCR4 interacts with CAF4 and CAF16LexA fusionB42 fusionβ-Galactosidase activityLineunits/mgLexA-CCR4-(1–837)B42301LexA-CCR4-(1–837)B42-CAF4542LexA-CCR4-(1–837)B42-CAF163703LexA-CCR4-(1–13/210–837)B4284LexA-CCR4-(1–13/210–837)B42-CAF4225LexA-CCR4-(1–13/210–837)B42-CAF166706LexA-CCR4-(1–395/420–837)B42-CAF4<27LexA-CCR4-(1–395/420–837)B42-CAF16<28LexAB42-CAF4<29LexAB42-CAF16<210LexA-CAF4B42-NOT1110011LexA-CAF4B42-DBF255012LexA-CAF4B422513LexA-CAF16B42-CAF166314LexA-CAF16B42<215LexA-CCR4-(1–837), B42-DBF2, and B42-NOT1 contain full-length versions of CCR4, DBF2, and NOT1 proteins, respectively. B42-CAF4 contains residues 545–659 of CAF4; B42-CAF16 contains residues 20–288 of CAF16; LexA-CAF4 contains residues 61–659 of CAF4; and LexA-CAF16 contains residues 20–288 of CAF16. All LexA proteins were LexA-(1–202) except for LexA-CAF16 which contained LexA-(1–87). β-Galactosidase activities were determined in strain EGY188 containing the p34 reporter (8 LexA operators upstream oflacZ, Ref. 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar) following growth on minimal medium lacking uracil, histidine, and tryptophan that contained 2% galactose, 2% raffinose. Assays repressed the average of at least four transformants, and S.E. was less than 20%. Open table in a new tab LexA-CCR4-(1–837), B42-DBF2, and B42-NOT1 contain full-length versions of CCR4, DBF2, and NOT1 proteins, respectively. B42-CAF4 contains residues 545–659 of CAF4; B42-CAF16 contains residues 20–288 of CAF16; LexA-CAF4 contains residues 61–659 of CAF4; and LexA-CAF16 contains residues 20–288 of CAF16. All LexA proteins were LexA-(1–202) except for LexA-CAF16 which contained LexA-(1–87). β-Galactosidase activities were determined in strain EGY188 containing the p34 reporter (8 LexA operators upstream oflacZ, Ref. 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar) following growth on minimal medium lacking uracil, histidine, and tryptophan that contained 2% galactose, 2% raffinose. Assays repressed the average of at least four transformants, and S.E. was less than 20%. In addition to two-hybrid interactions observed between CAF4 or CAF16 with CCR4, LexA-CAF4 was shown to interact specifically with B42-NOT1 and B42-DBF2 (Table II, lines 11 and 12). It should be noted that LexA-CAF4 was capable of activating transcription weakly by itself (line 13), a phenotype also observed with LexA-CCR4, LexA-CAF1, and several LexA-NOT proteins (3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar, 9Collart M.A. Struhl K. Genes Dev. 1994; 8: 525-537Crossref PubMed Scopus (173) Google Scholar, 13Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 487-495Crossref Scopus (96) Google Scholar, 11Oberholzer U. Collart M.A. Gene ( Amst. ). 1998; 207: 61-69Crossref PubMed Scopus (57) Google Scholar). To determine if the observed two-hybrid interactions were the result of in vivo physical association of CCR4 with CAF16 and CAF4, coimmunoprecipitation analysis was conducted. Immunoprecipitating CCR4 with anti-CCR4 antibody failed, however, to coimmunoprecipitate specifically B42-CAF16 or B42-CAF4 or their cognate unfused proteins (data not shown). These data suggest that CAF16 and CAF4 are not components of the 1-MDa CCR4-NOT complex since all of the NOT proteins and CAF1 can be immunoprecipitated with CCR4 in this core complex (1Liu H.-Y. Badarinarayana V. Audino D.C. Rappsilber J. Mann M. Denis C.L. EMBO J. 1998; 17: 1096-1106Crossref PubMed Scopus (173) Google Scholar, 3Draper M.P. Liu H.-Y. Nelsbach A.H. Mosley S.P. Denis C.L. Mol. Cell. Biol. 1994; 14: 4522-4531Crossref PubMed Scopus (73) Google Scholar, 4Bai Y. Salvadore C. Chiang Y.-C. Collart M.A. Liu H.-Y. Denis C.L. Cell Biol. 1999; 19: 6642-6651Google Scholar, 13Draper M.P. Salvadore C. Denis C.L. Mol. Cell. Biol. 1995; 15: 487-495Crossref Scopus (96) Google Scholar).1 Moreover, neither CAF16 nor CAF4 was found to be present in a purified 1-MDa CCR4-NOT complex.1 To determine whether the CAF16 and CAF4 proteins associated in the 1.9-MDa CCR4-NOT complex, gel filtration analysis was conducted. Following Superose 6 chromatography a subset of the CAF16 protein was found to migrate at 1.9 MDa, coincident with the size of the 1.9-MDa CCR4-NOT complex (Fig. 1, top panel). A similar analysis with CAF4 protein tagged with the HA1 epitope showed that CAF4-HA migrated at 1.9 MDa (Fig. 1, 2ndfrom top panel). Deletion of CCR4 was found to remove effectively CAF16 and CAF4 from the 1.9-MDa complex (Fig.1 , top two panels). A ccr4 deletion did not have this effect on CAF1 or CAF40 (another component of the 1-MDa CCR4-NOT complex) (Fig. 1, bottom two panels), nor did it have this effect on DBF2, which is also a presumed component of the 1.9-MDa CCR4-NOT complex (Fig. 1, middle panel). The effect of theccr4 deletion on CAF16 and CAF4 migration at 1.9 MDa supports the physical presence of CAF16 and CAF4 in the larger CCR4-NOT complex and that CCR4 is required for these proteins to associate in this complex. The observation that a majority of the CAF16 in the cell is not apparently in the 1.9-MDa complex suggests that CAF16 may have other functions than those dealing with CCR4 or that the CAF16 association with CCR4 is unstable. The above results suggest that CCR4 interacts with CAF16 and CAF4 but that CAF16 and CAF4 are external to the core 1-MDa CCR4-NOT complex. Since CAF1 is absolutely required for CCR4 association in the 1-MDa CCR4-NOT complex, we subsequently tested whether CCR4 interaction with CAF16 and CAF4 was dependent on CAF1. In a caf1 deletion strain, LexA-CCR4-(1–837) interacted with B42-CAF16 (280 units/mg β-galactosidase) as well as it did in a CAF1 strain (TableII, line 3; 370 units/mg β-galactosidase). These data confirm that CCR4-CAF16 interactions are separate from those that link CCR4 to the 1-MDa CCR4-NOT complex. A similar two-hybrid analysis with CAF4 showed that B42-CAF4 interacted with LexA-CCR4 in a caf1 strain (150 units/mg β-galactosidase) (compare with 54 units/mg β-galactosidase in the wild-type strain, Table II, line 2). The fact that CAF4 interacted better with CCR4 in a caf1 background suggests that CAF1 interferes with CAF4 binding to CCR4. Although CAF4 was not in the 1-MDa CCR4-NOT complex, we analyzed whether CAF4 when overexpressed was capable of immunoprecipitating with DBF2 and NOT1 based on their strong two-hybrid interactions (Table II, lines 11 and 12). Immunoprecipitating LexA-CAF4 with anti-LexA antibody was capable of coimmunoprecipitating B42-DBF2 (Fig.2, lane
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