A Conserved Domain of Yeast RNA Triphosphatase Flanking the Catalytic Core Regulates Self-association and Interaction with the Guanylyltransferase Component of the mRNA Capping Apparatus

Abstract

The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation. Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539. Sedimentation analysis indicates that purified Cet1(231–549)p is a homodimer. Cet1(231–549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231–549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276–549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p. An intermediate deletion protein Cet1(246–549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex. These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p. Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2μCET1(246–549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutationCET1(201–549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 othercet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression. Finally, we show that the in vivo function of Cet1(275–549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme. We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase. The 549-amino acid yeast RNA triphosphatase Cet1p catalyzes the first step in mRNA cap formation. Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that is dispensable for catalysis in vitro and for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is dispensable for catalysis but essential for Cet1p function in vivo; and (iii) a catalytic domain from residues 275 to 539. Sedimentation analysis indicates that purified Cet1(231–549)p is a homodimer. Cet1(231–549)p binds in vitro to the yeast RNA guanylyltransferase Ceg1p to form a 7.1 S complex that we surmise to be a trimer consisting of two molecules of Cet1(231–549)p and one molecule of Ceg1p. The more extensively truncated protein Cet1(276–549)p, which cannot support cell growth, sediments as a monomer and does not interact with Ceg1p. An intermediate deletion protein Cet1(246–549)p, which supports cell growth only when overexpressed, sediments principally as a discrete salt-stable 11.5 S homo-oligomeric complex. These data implicate the segment of Ceg1p from residues 230 to 275 in regulating self-association and in binding to Ceg1p. Genetic data support the existence of a Ceg1p-binding domain flanking the catalytic domain of Cet1p, to wit: (i) the ts growth phenotype of 2μCET1(246–549) is suppressed by overexpression of Ceg1p; (ii) a ts alanine cluster mutationCET1(201–549)/K250A-W251A is suppressed by overexpression of Ceg1p; and (iii) 15 othercet-ts alleles with missense changes mapping elsewhere in the protein are not suppressed by Ceg1p overexpression. Finally, we show that the in vivo function of Cet1(275–549)p is completely restored by fusion to the guanylyltransferase domain of the mouse capping enzyme. We hypothesize that the need for Ceg1p binding by yeast RNA triphosphatase can by bypassed when the triphosphatase catalytic domain is delivered to the RNA polymerase II elongation complex by linkage in cis to the mammalian guanylyltransferase. The Saccharomyces cerevisiae RNA 5′-triphosphatase Cet1p is an essential enzyme that catalyzes the first step of mRNA cap formation: the hydrolysis of the γ-phosphate of triphosphate-terminated pre-mRNA to form a diphosphate end that serves as the substrate for capping by RNA guanylyltransferase (1Tsukamoto 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 (79) Google Scholar, 2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Cet1p is the prototype of a newly identified family of divalent cation-dependent nucleoside triphosphatases that includes the RNA 5′-triphosphatases encoded by two families of DNA viruses, poxviruses and baculoviruses (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The enzyme family is defined by the presence of three conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids essential for triphosphatase activity (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Yu L. Shuman S. J. Virol. 1996; 70: 6162-6168Crossref PubMed Google Scholar, 5Yu L. Martins A. Deng L. Shuman S. J. Virol. 1997; 71: 9837-9843Crossref PubMed Google Scholar, 6Gross C.H. Shuman S. J. Virol. 1998; 72: 10020-10028Crossref PubMed Google Scholar, 7Jin J. Dong W. Guarino L.A. J. Virol. 1998; 72: 10011-10019Crossref PubMed Google Scholar). Motifs A–C of yeast Cet1p are located within the C-terminal half of the 549-amino acid protein (Fig. 1 A). The N-terminal 200-amino acid segment of Cet1p is dispensable for catalytic activity in vitro (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Moreover, the NΔ200 alleleCET1(201–549) is fully capable of supporting yeast cell growth (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Fungi and mammals have evolved different strategies to assemble bifunctional capping enzymes with triphosphatase and guanylyltransferase activities. In S. cerevisiae, separately encoded triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) proteins interact to form a heteromeric complex (1Tsukamoto 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 (79) Google Scholar, 2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar, 8Shibagaki Y. Itoh N. Yamada H. Nagata S. Mizumoto K. J. Biol. Chem. 1992; 267: 9521-9528Abstract Full Text PDF PubMed Google Scholar), whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked incis within a single polypeptide (Mce1p) (9Yue 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 (196) 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 (433) Google Scholar, 11Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 12Yamada-Okabe T. Doi R. Shimmi O. Arisawa M. Yamada-Okabe H. Nucleic Acids Res. 1998; 26: 1700-1706Crossref PubMed Scopus (41) Google Scholar, 13Tsukamoto T. Shibagaki Y. Murakoshi T. Suzuki M. Nakamura A. Gotoh H. Mizumoto K. Biochem. Biophys. Res. Commun. 1998; 243: 101-108Crossref PubMed Scopus (30) Google Scholar). Genetic evidence suggests that the physical linkage of the mammalian triphosphatase and guanylyltransferase domains is essential in vivo. For example, a lethal yeast cet1 deletion can be complemented by expression of MCE1 but only if the catalytically active mouse triphosphatase domain is linked incis to a catalytically active guanylyltransferase domain (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar,12Yamada-Okabe T. Doi R. Shimmi O. Arisawa M. Yamada-Okabe H. Nucleic Acids Res. 1998; 26: 1700-1706Crossref PubMed Scopus (41) Google Scholar). Our isolation of CET1 in a genetic screen for high copy suppressors of temperature-sensitive mutations of Ceg1p suggested that of the yeast triphosphatase and guanylyltransferase in be essential in vivo (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). of the of that be suppressed by Cet1p overexpression of within a C-terminal domain of Ceg1p (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar, E. T. S. Genes Dev. 1998; PubMed Scopus Google Scholar), as physical of a Ceg1p. of have to in vivo yeast in vitro (1Tsukamoto 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 (79) Google Scholar, E. T. S. Genes Dev. 1998; PubMed Scopus Google Scholar, T. T. M. Y. Arisawa M. Yamada-Okabe H. 1998; PubMed Scopus (30) Google Scholar). of is that the of the and the have not with a genetic of the function of a Ceg1p-binding Our to the of purified Cet1p with purified Ceg1p by (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). In the and of the as as the of complex as a in the are from the Moreover, the of the can be from the We have (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google that the N-terminal deletion a discrete complex with in that is to in a the catalytic domain and the Ceg1p-binding within which is fully in vivo. we physical and genetic analysis of the yeast RNA triphosphatase that includes the (i) of a domain of by (ii) by and C-terminal of as a domain for cell growth (iii) physical of the Cet1p N-terminal deletion and with Ceg1p; of of the temperature-sensitive growth phenotype of the deletion by overexpression of Ceg1p; of a lethal deletion which a enzyme with catalytic activity that is to interact with Ceg1p; and of the in vivo function of by fusion in cis with the guanylyltransferase domain of the mouse capping enzyme. Our that a protease-sensitive segment of Cet1p located to the catalytic domain self-association and with Ceg1p. of N-terminal deletion of CET1 by polymerase acid with that an and a in of the for and The with and and yeast (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). The to the amino acid of polypeptide C-terminal by that in of the for and and of the The with and of the alleles in is the of the from the and the yeast expression In the expression of the yeast RNA triphosphatase is the of the yeast The Cet1(246–549)p, and from and to and expression of the truncated proteins as (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). The proteins purified from by (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). The polypeptide of the by The Cet1p protein and by the with as the The enzyme a expression of in a as to the acid in to an N-terminal acid The in The to and and for by and in of to a of the for and for to a of and to the of the by for in a The to a of that with The with the and with of and The polypeptide of the by Ceg1p the and in the The The Ceg1p enzyme of the of truncated Cet1p proteins with and in of 1 the Ceg1p The for and a G. The in a for from the of the of by with of the protein for by with is the RNA of of a is the RNA triphosphatase and guanylyltransferase. of is the of CET1 which the and the of The CET1 in vitro by DNA The to (i) a of to the three (ii) (iii) The with and The CET1 DNA from and and separately and the E. in from from the These DNA yeast for and to the CET1 to of the to The which the CET1 and and to but not DNA from in vivo in E. and for the growth phenotype by In we a of of cet1 the by DNA The the acid RNA triphosphatase T. T. M. Y. Arisawa M. Yamada-Okabe H. 1998; PubMed Scopus (30) Google by from a DNA G. S. S. 1997; Scopus Google a that the and an that a of the The and and a yeast expression a of in which a the of The N-terminal deletion of by with that an and a in of the for The with and and to and In expression of the polypeptide is the of Genes fusion proteins of an N-terminal segment from yeast Cet1p and the catalytically active C-terminal guanylyltransferase domain of the mouse capping enzyme as The CET1 from residues and the CET1 as and an that to and an the C-terminal The with and the of to fusion of the in is the of the a N-terminal to with of and (Fig. analysis of the protein a polypeptide (Fig. to of of by to a that the N-terminal to that of the protein from the of the the triphosphatase of the expression in E. of of a with N-terminal from (Fig. A). to an polypeptide with N-terminal (Fig. A). two a polypeptide with and a with N-terminal from (Fig. A). These two the of the to of the protein of the protein to a cluster of of an whereas two The of the with suggested the of in 1 B. The is that the and are located in the N-terminal of by and to a polypeptide with N-terminal (Fig. to a with the (Fig. of to the of a with N-terminal (Fig. a with N-terminal when the the whereas the an with the (Fig. by a the N-terminal (Fig. the of the protein to a N-terminal and (Fig. The within are the in A. The of analysis is that the of to three are within a polypeptide segment from to We that segment is In the of the protein is and to a We that expression of the truncated a single copy the of the CET1 fully complemented the growth of a deletion cerevisiae, whereas expression of the more extensively truncated and not (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). In of the a domain of we a of N-terminal deletion alleles as The of the two alleles and the residues that by and The a as to the of the CET1 These a in which the CET1 and by of is the of a CET1 a is to acid is with a active CET1 a from (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). We that growth complemented by and but not by the more extensively truncated alleles (Fig. and a segment residues and that is for Cet1p function in vivo when the triphosphatase is from we that deletion of amino acids from the of Cet1p in of function in C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). we by a of C-terminal and that complemented growth of not not in which truncated to complemented not The N-terminal deletion of Cet1p in as and purified from by A). activity by hydrolysis (3Ho C.K. Pei Y. Shuman S. J. Biol. Chem. 1998; 273: 34151-34156Abstract Full Text Full Text PDF PubMed Scopus (74) Google as a function of enzyme. The of and to that of (Fig. in with the that Cet1p that active in vivo catalytic activity in that Cet1(246–549)p, which not growth, active in vitro The that triphosphatase function in vitro an N-terminal of the catalytic domain a only residues of catalytic A. We activity of deletion alleles in vivo be by expression of the the of a yeast we as as and expression is by the yeast These for function by the and the and to the to not Cet1(276–549)p, which is catalytically active in cannot cell growth when in vivo. The that with to and but not not the growth of the and in with CET1 The a phenotype the of (Fig. that growth of be complemented by by the The to and more the whereas and to the in vivo activity of are to cell growth We that overexpression of Cet1p from a suppressed mutations in the yeast guanylyltransferase Ceg1p. We that the ts guanylyltransferase for Cet1p and that the of Cet1p by (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). We have that the can be suppressed by but not by not These that deletion of the Cet1p segment from residues to with Ceg1p a that the ts phenotype of the the genetic enzymes can overexpression of the guanylyltransferase the growth of a of the we the with a and the growth of and with a CET1 and whereas with the only (Fig. the of a growth and as as as by (Fig. suppressed the ts growth phenotype of the These that is for with Ceg1p in vivo. In to the of the genetic the yeast triphosphatase and we a of alleles from of we of the CET1 to by The from and a expression in expression of the is the of the in vivo of yeast CET1 for growth by to the we of that for growth We identified that but to form DNA from and by for the ts cet1 DNA and (Fig. of the a single missense missense residues and alleles as mutations not in the changes different residues (Fig. of the mutations located within the essential triphosphatase motifs B, and of the changes within discrete of the domain of the the and cluster (Fig. of the alleles to growth be restored to the by with a the CET1 of be to growth the by with a the of the growth phenotype of yeast triphosphatase mutations by overexpression of guanylyltransferase for the The of of the N-terminal deletion of Cet1p by of the enzymes the we the of the Cet1p deletion with Ceg1p by the proteins in vitro and the a proteins and as in the polypeptide of the by The for is in A. The triphosphatase as a discrete the of the of the three a An S of for from the These that is a homodimer. that of an S of for (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google a to in a different The for the of and Ceg1p is in B. the yeast proteins form a discrete complex that sediments a and The of the complex suggested a that sediments as a whereas Ceg1p is consists of two molecules of and one molecule of Ceg1p. The for is in A. The protein as a single discrete a and The of suggests that is a that the triphosphatase activity with the polypeptide not The of in the presence of Ceg1p Ceg1p a discrete intermediate and (Fig. Ceg1p the when in a Ceg1p and not These show that Cet1(276–549)p, catalytically the to to the guanylyltransferase. We hypothesize that the of Ceg1p binding for the of to support yeast cell the self-association and Ceg1p-binding of yeast RNA triphosphatase from of intermediate deletion For example, Cet1(231–549)p by as a discrete S of A). with Ceg1p in the of a complex 7.1 S (Fig. the to the we complex to be a that the segment from residues to 230 of Ceg1p is not for Ceg1p binding in in the of the triphosphatase when the protein truncated from to The enzyme by two discrete as follows: a that and a of S A). of a can be of the and proteins and in the of the the of The of the of to of a suggests that the is a if not an of the The S is a homodimer. that that the triphosphatase activity the of the with the of the activity with the not The of the form of by the of the protein and the and These that the of the and and the of the polypeptide the two by to 1 not with Ceg1p in a of the to form a complex 7.1 S B, of the Ceg1p with the (Fig. B, The of the Ceg1p to proteins in the complex in the 7.1 S complex Finally, the of a of the of that by two discrete of S (Fig. and S (Fig. but the of the protein the with Ceg1p in the of a S complex (Fig. B, and the of Ceg1p with the The of the analysis can be as follows: (i) the C-terminal domain of Cet1p from residues to an to to form a which binds to Ceg1p; (ii) deletion of the segment from residues to a to form a salt-stable S consisting of Ceg1p and (iii) deletion of the segment from residues to 275 self-association and the of Cet1p to to Ceg1p. The a acid RNA triphosphatase T. T. M. Y. Arisawa M. Yamada-Okabe H. 1998; PubMed Scopus (30) Google Scholar). The C-terminal segment of from residues to to the of S. cerevisiae Cet1p from to residues and with In the N-terminal of and Cet1p are not We and a of N-terminal deletion alleles of the of the and by for of the S. cerevisiae The whereas lethal (Fig. to the in B, the and of to of and amino acids from the of Cet1p, whereas the lethal to a deletion of residues from These show that the of the two RNA triphosphatases are The S. cerevisiae deletion for growth in The whereas not The ts growth phenotype suppressed by the of a the S. the with to growth and high copy as the of S. cerevisiae The that in of the alleles a conserved that of the of yeast RNA triphosphatases to we two of and in which of amino acids within conserved by alanine cluster and conserved of the The residues are by in B. The a the of the CET1 and by for of a deletion The and and as as and In the and but not (Fig. The ts phenotype of the suppressed by the a (Fig. These implicate the as of a yeast RNA The in that expression of mammalian in yeast can the growth of a in a of of a CET1 of with a the bifunctional mouse capping of the yeast growth of which only the guanylyltransferase domain of mouse capping of (Fig. We the activity of two capping enzymes in which of the yeast RNA triphosphatase and are to the mouse guanylyltransferase capable of supporting growth of the (Fig. not The of is that the in for Cet1p residues the binding for yeast can be by the triphosphatase catalytic domain in cis to a guanylyltransferase. that the of We from that the segment from residues to is for Cet1p activity in a guanylyltransferase to catalytic activity have by the of the protein in (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). The to an for the and of the yeast capping and genetic evidence that the triphosphatase Cet1p consists of three domains as follows: (i) a 230-amino acid N-terminal segment that to catalysis and is dispensable for Cet1p function in vivo; (ii) a protease-sensitive segment from residues 230 to 275 that is essential for Cet1p function in vivo and that Cet1p self-association and Cet1p binding to the yeast guanylyltransferase Ceg1p; (iii) an essential C-terminal catalytic domain that includes the conserved triphosphatase motifs B, and of evidence that the of Cet1p with Ceg1p is the of in with N-terminal deletion of Cet1p to 275 with the of Ceg1p binding in the in vivo function of can be restored by protein in cis to mouse the need for the guanylyltransferase for the of a bifunctional enzyme in in yeast in cis as in mammals and the an conserved for the capping to the binding of the guanylyltransferase to the C-terminal domain of RNA polymerase II (9Yue 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 (196) 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 (433) Google Scholar, 11Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, E. T. S. Genes Dev. 1997; 11: PubMed Scopus Google Scholar). mouse capping enzyme and the C-terminal guanylyltransferase domain to the whereas the N-terminal mouse triphosphatase domain to the C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, C.K. Shuman S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In the guanylyltransferase binds to 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 (433) Google Scholar), whereas the yeast triphosphatase does not E. T. S. Genes Dev. 1998; PubMed Scopus Google Scholar). K. and S. In the guanylyltransferase the triphosphatase to the complex. is that the mammalian guanylyltransferase can as for the yeast Cet1p is completely in and catalytic from the mammalian RNA triphosphatase that is the mammalian triphosphatase and guanylyltransferase domains that is to the mammalian suggests that the mammalian guanylyltransferase can as a to proteins to the complex in vivo. The genetic Cet1p overexpression a in Ceg1p (2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar, E. T. S. Genes Dev. 1998; PubMed Scopus Google is by the that Ceg1p overexpression the phenotype of the The of is that is in with Ceg1p. analysis of the of that the is more complex. The form of the protein sediments as discrete salt-stable that does not interact in a with Ceg1p. the of does interact with Ceg1p in vivo phenotype of the as follows: (i) and the of is to function in with Ceg1p and yeast cannot (ii) expression of the of triphosphatase to interact with Ceg1p the for (iii) the be the that the of the for and expression of Ceg1p in vivo the from by a of active capping enzyme complex The in is to changes in the of the that the a in to a that does of Ceg1p suppressed the ts phenotype when the of in single copy from the CET1 of protein the (i) as the enzyme activity in and (ii) the protein as a discrete with of the and only of a of the in the S vivo of the are to be by the for the deletion we hypothesize that of the and the of Cet1p for Ceg1p and that Ceg1p overexpression can to a of the heteromeric complex. In we that with is not that the is Our is that of the S. cerevisiae RNA S. Cet1p suggests that the is conserved in RNA in the the and of the yeast complex. Itoh N. Mizumoto K. Y. J. Biol. Chem. Full Text PDF PubMed Google the of a bifunctional complex from yeast cell that of a guanylyltransferase and a RNA triphosphatase enzyme S in a with the a of and that the enzyme a N. Mizumoto K. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). An from yeast an of a guanylyltransferase and a RNA triphosphatase that S in a N. Yamada H. Y. Mizumoto K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that the triphosphatase to Cet1p, which an of but (1Tsukamoto 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 (79) Google Scholar, 2Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). We surmise that the triphosphatase in the enzyme purified is a of Cet1p the catalytic The form of guanylyltransferase is a a segment the B. Shuman S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Yamada-Okabe T. T. M. Y. Arisawa M. Yamada-Okabe H. 1998; PubMed Scopus (30) Google suggested that the S enzyme capping enzyme from yeast an of is an the analysis in the we that the enzyme complex in vitro from separately and Ceg1p proteins is a trimer and that by is a homodimer. An trimer for the capping enzyme complex the that (i) the binding of one Ceg1p to one of the of the the Ceg1p-binding of the (ii) triphosphatase to form a single