Homodimeric Quaternary Structure Is Required for the in Vivo Function and Thermal Stability of Saccharomyces cerevisiae and Schizosaccharomyces pombe RNA Triphosphatases
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303060200
ISSN1083-351X
AutoresStéphane Hausmann, Yi Pei, Stewart Shuman,
Tópico(s)RNA and protein synthesis mechanisms
ResumoSaccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic β-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain. Saccharomyces cerevisiae Cet1 and Schizosaccharomyces pombe Pct1 are the essential RNA triphosphatase components of the mRNA capping apparatus of budding and fission yeast, respectively. Cet1 and Pct1 share a baroque active site architecture and a homodimeric quaternary structure. The active site is located within a topologically closed hydrophilic β-barrel (the triphosphate tunnel) that rests on a globular core domain (the pedestal) composed of elements from both protomers of the homodimer. Earlier studies of the effects of alanine cluster mutations at the crystallographic dimer interface of Cet1 suggested that homodimerization is important for triphosphatase function in vivo, albeit not for catalysis. Here, we studied the effects of 14 single-alanine mutations on Cet1 activity and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface is lethal in vivo and renders Cet1 activity thermolabile at physiological temperatures in vitro. In addition, we identify individual residues within the pedestal domain (Ile470, Leu519, Ile520, Phe523, Leu524, and Ile530) that stabilize Cet1 in vivo and in vitro. In the case of Pct1, we show that dimerization depends on the peptide segment 41VPKIEMNFLN50 located immediately prior to the start of the Pct1 catalytic domain. Deletion of this peptide converts Pct1 into a catalytically active monomer that is defective in vivo in S. pombe and hypersensitive to thermal inactivation in vitro. Our findings suggest an explanation for the conservation of quaternary structure in fungal RNA triphosphatases, whereby the delicate tunnel architecture of the active site is stabilized by the homodimeric pedestal domain. RNA triphosphatase catalyzes the first step in mRNA cap formation, the cleavage of the β-γ phosphoanhydride bond of 5′-triphosphate RNA to yield a diphosphate end. In the second step of the pathway, the RNA diphosphate is capped with GMP by RNA guanylyltransferase to yield GpppRNA (1Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar). The budding yeast Saccharomyces cerevisiae encodes separate triphosphatase (Cet1; 549 aa 1The abbreviations used are: aa, amino acid(s); CTD, C-terminal domain; ts, temperature-sensitive; 5-FOA, 5-fluoroorotic acid; DTT, dithiothreitol; BSA, bovine serum albumin.) and guanylyltransferase (Ceg1; 459 aa) proteins that interact in trans to form a stable capping enzyme complex consisting of one Ceg1 protomer bound to a dimer of Cet1 (2Shibagaki Y. Itoh N. Yamada H. Hagata S. Mizumoto K. J. Biol. Chem. 1992; 267: 9521-9528Abstract Full Text PDF PubMed Google Scholar, 3Tsukamoto T. Shibagaki Y. Imajoh-Ohmi S. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1997; 239: 116-122Crossref PubMed Scopus (80) Google Scholar, 4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Although the fission yeast Schizosaccharomyces pombe also encodes separate triphosphatase (Pct1; 303 aa) and guanylyltransferase (Pce1; 402 aa) enzymes, they do not interact with each other (6Shuman S. Liu Y. Schwer B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12046-12050Crossref PubMed Scopus (96) Google Scholar, 7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 8Pei Y. Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The Cet1-Ceg1 interaction stabilizes the intrinsically labile guanylyltransferase activity of Ceg1 against thermal inactivation at physiological temperatures (9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). In addition, the physical tethering of Cet1 to Ceg1 facilitates recruitment of the triphosphatase to the RNA polymerase II elongation complex, via Ceg1 binding to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II (10McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (439) Google Scholar, 11Cho E. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (377) Google Scholar, 12Schroeder S.C. Schwer B. Shuman S. Bentley D. Genes Dev. 2000; 14: 2435-2440Crossref PubMed Scopus (309) Google Scholar, 13Komarnitsky P. Cho E. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (811) Google Scholar). Cet1 by itself does not interact with the CTD. In contrast, the S. pombe guanylyltransferase Pce1 is inherently thermostable, and its stability is unaffected by the presence of the triphosphatase Pct1 (9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Also, S. pombe employs a distinctive strategy of targeting capping to polymerase II transcripts, whereby the Pct1 and Pce1 enzymes bind independently to the phosphorylated CTD (8Pei Y. Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus, the fission yeast has elided on both counts the need for a triphosphatase-guanylyltransferase complex. It is therefore not surprising that Pct1 has no counterpart of the surface domain of Cet1 that mediates binding to the guanylyltransferase (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar). Although the fungal triphosphatase components display species-specific differences in their protein-protein interactions, they are nonetheless conserved with respect to their active site architecture, catalytic mechanism, and quaternary structure. The yeast triphosphatases belong to a family of metal-dependent phosphohydrolases that embraces the RNA triphosphatase components of the capping enzymes of unicellular eukaryotes and certain DNA viruses (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 14Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Shuman S. Nat. Rev. Mol. Cell Biol. 2002; 3: 619-625Crossref PubMed Scopus (116) Google Scholar). The family is defined by the presence of two conserved glutamate-containing motifs (β1 and β11 in Fig. 1) and the signature property of hydrolyzing NTPs to NDPs in the presence of manganese or cobalt (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar,14Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The crystal structure of the S. cerevisiae RNA triphosphatase Cet1 revealed that the enzyme is a homodimer with active sites located within parallel topologically closed tunnels composed of eight β strands (16Lima C. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) (Fig. 1). The “triphosphate tunnel” architecture is supported by an intricate network of hydrogen bonds and electrostatic interactions within the cavity, most of which are required for catalytic activity (17Pei Y. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 1999; 274: 28865-28874Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar,18Bisaillon M. Shuman S. J. Biol. Chem. 2001; 276: 17261-17266Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The tunnel floor rests on a globular “pedestal” domain. Amino acid sequence comparisons and mutational analyses of the RNA triphosphatases from other fungi (e.g. Candida albicans and S. pombe), microsporidia, protozoa, and Chlorella virus underscore the conservation of the β strands that comprise the triphosphate tunnel (15Shuman S. Nat. Rev. Mol. Cell Biol. 2002; 3: 619-625Crossref PubMed Scopus (116) Google Scholar). Mutational analyses of the C. albicans and S. pombe RNA triphosphatases indicate that their active sites and catalytic mechanism adhere closely to that of Cet1 (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 19Pei Y. Lehman K. Tian L. Shuman S. Nucleic Acids Res. 2000; 28: 1885-1892Crossref PubMed Scopus (37) Google Scholar). The S. cerevisiae and S. pombe RNA triphosphatases are both homodimers (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar, 16Lima C. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Available evidence indicates that homodimer formation is essential for Cet1 function in vivo but not for catalytic activity. Deletion analysis showed that the C-terminal domain Cet1(276–549) has a monomeric quaternary structure and retains activity in vitro (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, the monomeric domain by itself cannot support yeast cell growth, even when it is overexpressed at high gene dosage under the control of a strong promoter. Interpretation of the deletion data is complicated by the fact that an N-terminal truncation to position 275 also removes the guanylyltransferase-binding site 247WAQKW251, which is located on the protein surface (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 16Lima C. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Ho C.K. Lehman K. Shuman S. Nucleic Acids. Res. 1999; 27: 4671-4678Crossref PubMed Scopus (31) Google Scholar, 21Takase Y. Takagi T. Komarnitsky P.B. Buratowski S Mol. Cell. Biol. 2000; 20: 9307-9316Crossref PubMed Scopus (25) Google Scholar) and is responsible for Cet1-mediated stabilization of the guanylyltransferase Ceg1 (9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The in vivo function of Cet1(276–549) is restored when the monomeric triphosphatase is fused to either S. pombe guanylyltransferase (Pce1) or the guanylyltransferase domain of mammalian capping enzyme (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The S. pombe and mammalian guanylyltransferases bind to the phosphorylated CTD (8Pei Y. Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 10McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (439) Google Scholar, 22Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12898-12903Crossref PubMed Scopus (199) Google Scholar, 23Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and can thereby act as vehicles to deliver the fused monomeric yeast RNA triphosphatase to the RNA polymerase II elongation complex (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Also, because the S. pombe and mammalian guanylyltransferases are thermostable (unlike Ceg1), the chimeric capping enzymes bypass the need for the Ceg1-stabilization function of the 247WAQKW251 peptide of Cet1 (9Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 36116-36124Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). To focus specifically on the role of homodimerization in Cet1 function in vivo, we previously performed an alanine cluster mutational analysis guided by the Cet1 crystal structure (24Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Double-alanine mutations at vicinal amino acids were introduced into the biologically active protein Cet1(201–549), which contains both the guanylyltransferase-binding and catalytic domains. A total of 42 residues were changed to alanine, 24 of which were constituents of the crystallographic dimer interface. Four of the Ala cluster alleles were lethal in vivo. Three other Ala cluster mutants displayed temperature-sensitive (ts) growth defects, even when the mutant alleles were present in high copy under the control of a strong promoter. Several of the lethal and ts mutations were suppressed by fusion of the Cet1-Ala/Ala protein to the mammalian guanylyltransferase. Moreover, two of the lethal mutant proteins were characterized in vitro and found to be catalytically active monomers (24Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). These results indicated that homodimerization of the budding yeast RNA triphosphatase is critical in vivo when Cet1 functions in concert with the endogenous yeast guanylyltransferase. It remains unclear why homodimerization of yeast RNA triphosphatase is important in vivo. If the dimer is critical for the functional interactions of S. cerevisiae RNA triphosphatase and RNA guanylyltransferase, then it is not obvious why a homodimeric quaternary structure for RNA triphosphatase would be conserved in the fission yeast S. pombe, where the triphosphatase and guanylyltransferase components do not interact physically. On the other hand, homodimerization may confer added value to the triphosphatase in other ways that are independent of the guanylyltransferase component of the capping apparatus. Here we conduct a series of experiments to define the individual essential constituents of the Cet1 homodimer interface and probe the role of quaternary structure in triphosphatase function in vivo and in vitro. Guided by the initial results of the Ala cluster mutagenesis, we tested the effects of 14 single-alanine mutations on Cet1 activity in vivo and thereby pinpointed Asp280 as a critical side chain required for dimer formation. We find that disruption of the dimer interface renders Cet1 thermolabile in vitro. We engineered a catalytically active monomeric version of S. pombe Pct1 and show that it is thermolabile in vitro. Introduction of the monomeric triphosphatase into a S. pombe pct1Δ strain confers a dosage-suppressible lethal phenotype in vivo. We propose a model whereby homodimerization of the globular pedestal domain is critical to stabilize the delicate active site tunnel architecture of the fungal RNA triphosphatases. Mutagenesis of S. cerevisiae RNA Triphosphatase—Alanine mutations were introduced into the CET1(201–549) gene by PCR (25Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6986) Google Scholar). The mutated genes were inserted into the yeast CEN TRP1 plasmid pCET1–5′3′, where expression of the inserted gene is under the control of the natural CET1 promoter (26Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). The inserts were sequenced completely to exclude the acquisition of unwanted mutations during amplification and cloning. The in vivo activity of the mutated CET1 alleles was tested by plasmid shuffle. Yeast strain YBS20 (trp1 ura3 leu2 cet1::LEU2 p360-Cet1[CEN URA3 CET1]) was transformed with TRP1 plasmids containing the wild-type and mutant alleles of CET1(201–549). Trp+ isolates were selected and then streaked on agar plates containing 0.75 mg/ml 5-fluoroorotic acid (5-FOA). Growth was scored after 7 days of incubation at 18°, 25°, 30°, and 37 °C. Lethal mutants were those that failed to form colonies on 5-FOA at any temperature. Individual colonies of the viable CET1 mutants were picked from the FOA plate at permissive temperature and patched to YPD agar. Two isolates of each mutant were tested for growth on YPD agar at 18°, 25°, 30°, and 37 °C. Growth was assessed as follows: +++ indicates colony size indistinguishable from strains bearing wild-type CET1(201–549); ++ denotes slightly reduced colony size; + indicates that only pinpoint colonies were formed. Purification of Recombinant S. cerevisiae Triphosphatase—NdeI/BamHI fragments encoding mutated versions of Cet1(201–549) were excised from the respective pCET1–5′3′ plasmids and inserted into pET16b. Wild-type Cet1(201–549) and the Cet1(201–549)-Ala mutants were expressed in Escherichia coli BL21(DE3) at 18 °C by isopropyl-1-thio-β-d-galactopyranoside induction for 20 h in the presence of 2% ethanol (14Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The proteins were purified from soluble bacterial lysates by nickel-agarose chromatography as described previously (4Lehman K. Schwer B. Ho C.K. Rouzankina I. Shuman S. J. Biol. Chem. 1999; 274: 22668-22678Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The 0.2 m imidazole eluate fractions containing Cet1(201–549) were dialyzed against 50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 2 mm DTT, 10% glycerol, 0.05% Triton X-100, then stored at –80 °C. Mutagenesis of S. pombe RNA Triphosphatase—Gene fragments encoding N-terminal-truncated versions of Pct1 were generated by PCR amplification using sense primers that introduced an NdeI site at the codons for amino acids 41 or 51. The antisense primers introduced a BamHI site immediately downstream of the stop codon. The PCR products were digested with NdeI and BamHI and then inserted into pET16b. Full-length Pct1 and the NΔ40 and NΔ50 mutants were produced in E. coli as N-terminal His10-tagged fusions and purified from soluble bacterial lysates by nickel-agarose chromatography as described previously (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar,8Pei Y. Hausmann S. Ho C.K. Schwer B. Shuman S. J. Biol. Chem. 2001; 276: 28075-28082Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Glycerol Gradient Sedimentation—Aliquots (45 μg) of the nickel-agarose preparations of wild-type Cet1(201–549) and the D280A mutant were mixed with BSA (40 μg) and cytochrome c (40 μg) in 0.2 ml of buffer G (50 mm Tris HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 2 mm DTT, 0.05% Triton X-100). Aliquots (50 μg) of the nickel-agarose preparations of wild-type Pct1 and the NΔ40 and NΔ50 mutants were mixed with BSA (50 μg), ovalbumin (50 μg), and cytochrome c (50 μg) in 0.2 ml of buffer G. The mixtures were layered onto 4.8 ml of 15–30% glycerol gradients containing buffer G. The gradients were centrifuged in a Beckman SW50 rotor at 50,000 rpm for 24 h at 4 °C. Fractions (∼0.2 ml) were collected from the bottoms of the tubes. Aliquots (20 μl) of odd-numbered fractions were analyzed by SDS-PAGE. Polypeptides were visualized by staining with Coomassie Blue dye. Assay of Triphosphatase Activity—Reaction mixtures (10 μl) containing 50 mm Tris HCl (pH 7.5), 5 mm DTT, 2 mm MnCl2,1mm [γ-32P]ATP, and Cet1 or Pct1 as specified were incubated for 15 min at 30 °C. The reactions were quenched by adding 2.5 μl of 5 m formic acid. An aliquot of the mixture was applied to a polyethyleneimine-cellulose TLC plate, which was developed with 0.5 m LiCl and 1 m formic acid. The release of 32Pi from [γ-32P]ATP was quantitated by scanning the TLC plate with a PhosphorImager. Test of RNA Triphosphatase Function in Vivo in S. pombe—The full-length pct1 + and truncated pct1-NΔ40 and pct1-NΔ50 cDNAs were cloned into the S. pombe expression vectors pREP81X, pREP41X, and pREP3X (LEU2 ars1 +) so as to place them under the control of the nmt1**, nmt1*, and nmt1 promoters, respectively (27Basi G. Schmmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (580) Google Scholar, 28Forsburg S.L. Nucleic Acids Res. 1993; 21: 2955-2956Crossref PubMed Scopus (403) Google Scholar). The plasmids were transformed into a heterozygous pct1 + /pct1::kanMX diploid (29Pei Y. Schwer B. Saiz J. Fisher R.P. Shuman S. BMC Microbiol. 2001; 1: 29Crossref PubMed Scopus (15) Google Scholar) using the lithium acetate method (30Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2932) Google Scholar). The Leu+ diploid transformants were selected and then sporulated on ME plates at room temperature. A loopful of cells was inoculated into 500 μl of sterile water, and the mixture was incubated overnight at 28 °C with 10 μl of β-glucuronidase (Sigma G7770). The spores were plated on EMM(-Leu) agar medium and incubated at 30 °C. Individual colonies were then restreaked onto YES agar and on YES agar containing 200 μg/ml G418. Growth was scored after incubation for 5–7 days at 30° and 37 °C. Structure-based Mutational Analysis of the Cet1 Homodimer Interface—The Cet1 homodimer interface is extensive, with a buried surface area of 1860 Å2 per protomer (16Lima C. Wang L.K. Shuman S. Cell. 1999; 99: 533-543Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Elements that comprise the dimer interface are strands β2 and β3, helices α1 and α4, the loop immediately preceding α1, the loop between β9 and β10, and the loop between α3 and α4 (see Fig. 1). The molecular contacts of the dimer interface entail multiple hydrophobic interactions and a network of side-chain and main-chain hydrogen bonds. We previously tested the effects of 21 double-alanine mutations of vicinal amino acids on Cet1 function in vivo (24Lehman K. Ho C.K. Shuman S. J. Biol. Chem. 2001; 276: 14996-15002Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Twenty-four of the mutated residues were constituents of the crystallographic dimer interface. The Ala cluster mutations also targeted residues in helices α1 and α4 that compose the hydrophobic core of the pedestal upon which the triphosphate tunnel rests. Four of the Ala cluster alleles were lethal in vivo: D279A-D280A, C330A-V331A, L519A-I520A, and F523A-L524A. Three Ala cluster mutants displayed temperature-sensitive (ts) growth defects, even at high gene dosage: F272A-L273A, I470A-I472A, and I529A-I530A. The cluster mutagenesis approach provided useful information in two respects: (i) it showed that 15 of the amino acids that comprise the crystallographic homodimer interface are not important for Cet1 function in vivo, and (ii) it narrowed down the critical constituents of the homodimer interface to one or both of the side chains of the clusters at which Ala-Ala mutations elicited severe growth defects. Here we set out to identify which individual components of the dimer interface are functionally relevant, by testing the effects of single-alanine mutations at each of the residues of those clusters. As in previous studies, the mutations were introduced into the biologically active Cet1(201–549) protein. The CET1(201–549)-Ala genes were cloned into a CEN TRP1 vector under the control of the natural CET1 promoter and then tested by plasmid shuffle for their ability to complement a cet1Δ strain of S. cerevisiae. Growth of cet1Δ is contingent upon maintenance of a wild-type CET1 allele on a CEN URA3 plasmid. Therefore, the cet1Δ strain is unable to grow on agar medium containing 5-FOA (5-fluoroorotic acid, a drug that selects against the URA3 plasmid) unless it is first transformed with a biologically active RNA triphosphatase gene on the TRP1 plasmid. Trp+ CET1(201–549)-Ala transformants were tested for growth on 5-FOA. The results are summarized in Table I. Triphosphatase mutations were judged to be lethal if they failed to support colony formation on 5-FOA after prolonged incubation at four different temperatures (18°, 25°, 30°, and 37 °C). Two of the single-Ala mutations were lethal by this criterion: D280A and I520A.Table IMutational effects on CetI function in vivoCET1 mutantGrowthCross-dimer contactsIntra-protomer contacts18°C25°C30°C37°CF272A+++++++++++Ile470 Ile529 Ile530L273A++++++++++++Tyr354 Ile358 Ile529Phe272 Ile275D279A++++++++++++D280A----Gln329(N) Arg531 Arg532C330A++++++++++++Val326 Ser328 Cys330Val326V331A+++++++++++Ser327 Ser328Leu415 Leu417I470A++++++--Phe272Ile355 Leu495I472A+++++++++++Phe348 Ile355L519A++++++++++Ile268Cys467 Ile497 Leu502I520A----Val285 Val289 Tyr516 Leu524F523A+++++--Met308 Ile428 Ile497L524A++++++--Val285 Ile428 Ile520I529A++++++++++++Phe272 Leu273 Pro277I530A+++++--Phe272Phe310 Val493 Leu495 Open table in a new tab The other 12 mutants gave rise to 5-FOA-resistant colonies at one or more of the selection temperatures. The viable CET1(201–549)-Ala strains were then tested for growth on rich medium (YPD agar) at 18°, 25°, 30°, and 37 °C. A hierarchy of severity of mutational effects was thereby revealed (Table I). Four of the mutants displayed a tight ts phenotype: I470A and L524A cells grew as well as wild-type yeast at 18° and 25 °C (scored as +++ based on colony size) but failed to grow at 30° and 37 °C (–growth); F523A and I530A cells grew normally at 18 °C but formed small colonies at 25 °C (scored as ++) and no colonies at 30° and 37 °C. Four of the mutants displayed weaker ts phenotypes: the F272A, V331A, and I472A strains grew normally at 18°, 25°, and 30 °C but formed small colonies at 37 °C; L519A cells grew normally at 18°, 25°, and 30 °C but formed only pinpoint colonies at 37 °C. Four of the mutants, L273A, D279A, C330A, and I529A, grew as well as wild-type yeast at all temperatures tested. Three of the residues at which single-alanine substitutions elicited significant growth defects are components of the crystallographic homodimer interface: Asp280, Ile470, and Ile530. The Asp280 side chain is the only one of these that is strictly essential for cell viability. Thus, Asp280 appears to be an important constituent of the homodimer interface. None of the other three amino acids (Ile520, Phe523, and Leu524) at which alanine mutations resulted in significant growth defects are involved in cross-dimer contacts; rather, they are components of the hydrophobic core of the pedestal domains of the individual protomers. Biochemical Characterization of Mutant Enzymes—We produced the 14 Cet1(201–549)-Ala proteins in bacteria as His10-tagged fusions and purified them from soluble bacterial lysates by nickel-agarose chromatography. Wild-type His10-Cet1(201–549) was purified in parallel. SDS-PAGE analysis showed that the ∼44-kDa Cet1(201–549) protein was the predominant species in each enzyme preparation (Fig. 2A). Phosphohydrolase activity was assayed by the release of 32Pi from [γ-32P]ATP in the presence of manganese chloride (7Pei Y. Schwer B. Hausmann S. Shuman S. Nucleic Acids Res. 2001; 29: 387-396Crossref PubMed Google Scholar). The extents of ATP hydrolysis increased as a function of input enzyme for each protein (Fig. 2B). A specific activity for the wild-type Cet1(201–549) of 0.31 nmol of ATP hydrolyzed per n
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