Molecular Cloning and Sequencing of a cDNA Encoding N

Vol. 264, No. 21. Issue of July 25, PP. 12339-12343, 1989 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q

1989 by The American Society for Biochemistry and Molecular Biology. Inc.

Molecular Cloning and Sequencing of a cDNA Encoding N"-Acetyltransferasefrom Saccharomyces cereuisiae* (Received for publication, March 9, 1989)

Fang-Jen S. Lee$§, Lee-Wen Lin$§, and John A. Smith$ll From the Departments of $Molecular Biology and llf'athology, Massachusetts General Hospital and the §Departments of Genetics and Pathology, Haruard Medical School, Boston,Massachusetts 02114

Acetylation is the most frequently occurring chemical modification of the a-NHngroup of eukaryoticproteins and is catalyzed by an No-acetyltransferase.Recently, a eukaryotic Nu-acetyltransferase waspurified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J. S . , Lin L.-W., and Smith, J. A. (1988) J. Biol. Chem. 263,14948-14955). This articledescribes the cloning from a yeast Xgt 11 cDNA library and sequencing of a full length cDNA encoding yeast N"acetyltransferase. DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called A A A l , amino-terminal, a-amino, acetyltransferase) is present as a single copy located on chromosome IV. The use of this cDNA will allow the molecular details of the role of Nu-acetylation in the sortingand degradation of eukaryotic proteins to be determined.

Amino-terminal acylation is an important co- and posttranslational modification of proteins in prokaryotic and eukaryotic cells, Although formyl, pyruvoyl, a-ketobutyryl, glycosyl, glucuronyl, a-aminoacyl, p-glutamyl, myristoyl, and acetyl are well known N"-acylating groups, it is clear that acetylation is the most common chemical modification of the a-NH2 group of eukaryotic proteins (reviewed in Refs. 1 and 2). Acetylation is mediated by at least one Ne-acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the cu-NH2 group of proteins and peptides. Since the first identification that an acetyl moiety was the amino-terminal blocking group of tobacco mosaic virus coat protein (3) and a-melanocyte-stimulating peptide (4), a large number of proteins from various organisms have been shown to possess acetylatedamino-terminal residues. For example, mouse L-cells and Ehrlich ascites cells have about 80% of their intracellular soluble proteins N"-acetylated (5, 6). Ne-Acetylation plays a role in normal eukaryotic translation and processing (7) and protects against proteolytic degradation (8,9). Further, the rateof protein turnover mediated by the ubiquitin-dependent degradation system depends on the presence of a free a-NH, group at theamino terminus of model proteins (IO, II), and this dependence indicates that N"-acetylation plays a crucial role in impeding protein turnover. * This work was supported by a grant from the Hoechst Aktiengesellschaft (Frankfurt/Main, West Germany). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. hns been submitted The nucleotide sequence(s)reported in this paper to the GenBankTM/EMBL Data Bank withaccessionnumberfs) M23166.

Although the presence of N"-acetyltransferases had previously been demonstrated in Escherichia coli (121, rat liver (13-15), rat brain (16), rat pituitary (17-20), bovine pituitary (19), bovine lens (21), hen oviduct (22), and wheat germ (231, enzyme from these sources was never purified more than 40fold. Recently, an N"-acetyltransferase from Saccharomyces cereuisiae was purified to apparent homogeneity (4600-fold) and characterized as a dimeric protein, whose subunit M , was 95,000 and which would effectively transfer an acetyl group to various synthetic peptide substrates (including ACTH (124),' human superoxide dismutase (l-24), and yeast alcohol dehydrogenase (1-24) (24). Further, it was demonstrated that this enzyme would not transfer anacetyl group to the €-amino group of lysyl residues in various peptide substratesand histones. This paperreports the complete amino acid sequence for the yeast Ne-acetyltransferase deduced from cDNA analysis, demonstrates that theenzyme is encoded by a single gene (AAA1,amino-terminal, a-amino, acetyltransferase), and localizes this gene to chromosome IV. This yeast cDNA forms the basis for elucidating the biological function and regulation of Nu-acetylation in eukaryotic protein synthesis and degradation. EXPERIMENTAL PROCEDURES' RESULTS

Analyses of Tryptic Peptides of N'-Acetyltransferuse-The method adopted for the identification and cloning of cDNA sequences derived from N"-acetyltransferase mRNA utilized oligonucleotide probes that were constructed based on amino acid sequences of purified N"-acetyltransferase tryptic peptides and on codon-usage frequency data (34). Tryptic peptides from yeast Nu-acetyltransferase were separated by reversed-phase HPLC and collected as 62 pools representing distinct peaks or shoulders (Fig. 1). After one or two additional isocratic HPLC separations to resolve further individual sequenceable tryptic peptides (25), the sequences of 16peptides, comprising approximately 30% of the entire N"-acetyltransferase molecule, were determined. Synthesis of Oligonucleotide Probes-Two synthetic, codonusage based oligonucleotides of 57 bases (N1) and 48 bases (N2) was prepared based on the sequences of a 19-residue The abbreviations used are: ACTH, adrenocorticotropic hormone; HPLC, high performance liquid chromatography; NaPP,, sodium pyrophosphate; PEG, polyethylene glycol; R, A or G; SDS, sodium dodecyl sulfate; TPCK, L-l-tosylamido-2-phenylmethyl chloromethyl ketone; X, G or A or C or T; Y, C or T; Pth, phenylthlohydantoin; PAGE, polyacrylamide gel electrophoresis. Portions of this paper (including "Experimental Procedures" and Figs. 1 and 3-6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

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Molecular Cloning and Sequencing of a cDNA Encoding N*-Acetyltransferase

(peptide 27-3) and a 16-residue (peptide 11-3-2) peptide and the known codon-usage frequencies (34) (Fig. 2 A ) . In addition, two degenerate oligonucleotide probes of 23 (5' CCXTTGACYTTYTTRCAAGAYAA 3') and 20 (3' TCRTCRTGCATYTGRAARTA 5 ' ) bases with 64- and 32-fold redundancy, designated N3 and N4, were synthesized based on sequence data for peptides 29-1 and 10-3-1, respectively. Cloning of the Yeast N"-Acetyltransferase cDNA-After initial screening of 500,000 recombinant cDNA clones in the yeast Xgtll cDNA library, 11 clones were detected which hybridized to both oligonucleotides N 1 and N2. These clones,

designated AN1 to XN11, also hybridized with oligonucleotide N3 andN4, and theircDNA inserts were analyzed by restriction enzyme digestions and DNA blot analyses. The use of four oligonucleotide probes derived from four discrete amino acid sequences allowed the unequivocal identification of the cDNA clones encoding N"-acetyltransferase. EcoRI digestion revealed inserts that lacked internal EcoRI sites and ranged from 2.0 to 2.7 kilobase pairs. The six longest cDNA inserts were subcloned as EcoRI fragments into the Bluescript plasmid, and additional restriction enzyme mapping, DNA blot analyses, and nucleotide sequence analyses were carried out.

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FIG. 2. Cloning and sequencing of the cDNA encoding yeast N"-acetyltransferase.A, oligonucleotide probes used for initially screening the Xgtll library. The amino acid sequences of two tryptic peptides were used to construct the codon-usage frequency based oligonucleotide probes. The nucleotide positions indicated by the asterisks differ from the actual DNA sequence shown in C. The numbering of the tryptic peptides is as follows: the first number refers to the corresponding peak in Fig. 1, the second number refers to the peak in the first isocratic HPLC separation (data not shown), and the third number refers to the peak in the second isocratic HPLC separation (data not shown). B, restriction map and DNA sequencing strategy for the cDNA clones. The arrows indicate the direction and extent of sequence determination for each fragment after exonuclease I11 deletion. C, nucleotide and deduced amino acid sequence of Nu-acetyltransferase cDNA clones. The amino acid sequences of HPLC-purified tryptic peptides determined by automated protein sequence analysis are also shown. The protein sequence analyses were completed with repetitive yields between 87 and 93% for 100-200 pmol of each peptide.

Molecular Cloning

and Sequencing of a cDNA Encoding Ne-Acetyltransferase

All six cDNA clones (pBN1, pBN3, pBN7, pBN9, pBN10, and pBN11)displayed identical restriction maps (Fig. 2B). Sequence Analysis of the cDNA Clones-The complete nucleotide sequence,both orientations,of pBNl was determined using exonuclease 111 deletions and the double-stranded dideoxy chain termination method. The cDNA contained sequences encoding all 16 tryptic peptides but in two different reading frames,which could be aligned by the insertion of a n additional base between residue 1409 and 1411. Hence, the nucleotide sequences within the corresponding region of the other 5 cDNA clones were determined using synthetic oligonucleotide primers. These 5 clones each contained an additional T a t nucleotide 1410 which resulted ina singleextended open reading frame. It is likely that the deletion of a T at nucleotide 1410 in clone p B N l is an artifact of the cDNA synthesis. The complete nucleotide sequence of the yeast Ne-acetyltransferase cDNA is shown in Fig. 2C and contains a long open reading frame extending from nucleotide 22 to 2583, because there are no methionineresidues between the beginning of the open reading frame and the first tryptic peptide identified and because an in-frame terminationcodon (nucleotides 13-15) closely precedes the proposed site of initiation. The cDNA contains a nonstandard polyadenylationsignal (ATAAAA) located 18 nucleotides upstream from the poly(A) tail. RNA, DNA, and Chromosomal Blot Analyses-RNA blot analysis of yeast poly(A)+ mRNA using a random-primed, 32P-labeled cDNA probe (pBN1) revealed a 2.7-kilobase pair RNA band (Fig. 3). DNA blot analysis of restriction enzymedigested yeast genomic DNA (Fig. 4) with the same probe revealed afragment pattern consistent with a singlecopy gene encoding the sequences present in thecDNA (Fig. 2B). Chromosomal analysis with the same probe indicated that the yeast NO-acetyltransferase gene ( A A A I ) is located on chromosome IV (Fig. 5). Comparison of DNA and Protein Sequence Data for Yeast NO-Acetyltransferase cDNA with DNA and Protein Sequence Databases-The EMBL Nucleic Acid Database revealed an 842-base identity between a 3' region of the cDNA encoding N"-acetyltransferase, beginning a t nucleotide 1858 and ending at nucleotide 2699, and thegenomic DNA sequence upstream of the 5' end of the SIR2gene located on chromosome 4 (45): Comparisons between the protein sequence of yeast Nu-acetyltransferase and choline acetyltransferase fromDrosophilia (46), chloramphenicol acetyltransferase (47), and two ribosomal N"-acetyltransferases ( R i d and R i d ) from E. coli (48), and arylamine acetyltransferase from chickenliver (49) revealeda percentsimilarity of 10, 12, 12, 14, and 15%, respectively, although there are sequences of 6 to 16 amino acid residues which have percent similarities between 44 and 83% (50). Hydrophobicity Profile for Yeast N*-Acetyltransferase-The hydrophobicity profile was determinedusing the algorithmof Kyte and Doolittle(51) with a window size of 9 (Fig. 6). The protein is rich in charged amino acids, including 96 lysine, 37 arginine, 59 aspartic acid, and 60 glutamic acid residues. In addition, there is an extended hydrophilic region between residues 508 and 720, which is rich in residues associated with P-turn conformations (52). DISCUSSION

This is the first report of the molecular cloning and complete cDNAsequence analysis of a eukaryotic N"-acetyltransD. Shore, M. Squire, and K. A. Nasmyth,unpublished deposited in the EMBL Nucleic Acid Database.

data

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ferase gene, however, a cDNA for a P-endorphin-specific Neacetyltransferase cDNA was previouslyidentified in a rat brain Xgtll library, although it was not sequenced (53). The yeastNu-acetyltransferaseprotein is encoded by anopen reading frame of 2562 bases and consistsof 854 amino acids. The molecular weight calculated from the amino acid composition is 98,575, whichagrees well with the previously determined subunit M, of 95,000 +_ 2,000 (24). Sinceprevious protein sequence analysis of the native proteinrevealed it to be amino-terminally blocked (24), itis likely that the mature protein lacks the amino-terminal Met residue and is acylated (possibly acetylated) at the penultimate seryl residue (1, 2, 54-56). Although the enzyme is not known to be a glycoprotein, it contains six putative N-glycosylation sites (i.e. AsnX-Ser (or Thr) sequences) at residues 120-122,161-163,643645, 702-704,761-763,792-793. The extended, hydrophilic region between residues 508 and 720 is an unusual structural feature of the molecule, although it i s not clear whether this region plays a functional role inthe regulation orlocalization of the enzyme. A comparison of the protein sequences of NOacetyltransferase to other acetyltransferases does not reveal an appreciable percentsimilarity between them,although certain short sequences have a greater than 50% similarity. These are likely regions where site-specific mutations should be introduced in early attempts to identifyresidues involved in catalysis. Taken together, the Northern and Southern blots indicate that there is one gene encoding this NO-acetyltransferase. However, it is not clear whether or not yeast contains still otheracetyltransferases capable of modifying thea-NH2 group of proteins. Further, previous studies on the substrate specificity of the yeast N"-acetyltransferase have clearly demonstrated that this enzyme is not capable of acetylating tNH, groups in peptide substrates or in histones, although a histone-specific acetyltransferase has been demonstrated in yeast (57). The A A A l gene is located on chromosome 4 and is positioned immediately upstream of the SIR2 gene, which mediates trans repression of the transcriptiono f the mating type HMR and HML loci. At present, there is no clear-cut relationship between the function of these genes and AAAl. This cDNA forms the basis for elucidating the biological function and regulation of Na-acetylation in protein synthesis and degradation. Further,by using site-directed mutagenesis, the catalytic mechanism and regulation of W-acetyltransferase can be determined. Acknowledgements-We thank Dr. Jen Sheen for her help with the construction of the yeast cDNA library. We thank Dr. Brian Seed for his critical reading of the manuscript. We also wish to acknowledge J. the skilled technicalassistance of PaulGallantandThomas McLaughlin. REFERENCES 1. Tsunasawa, S., and Sakiyama, F. (1984) Methods Enzymol. 106,

165-170 2. Driessen, H. P. C., De Jong, W.W., Tesser, G. I., and Bloemendal, H. (1985) CRC Crit. Reu. Biochem. 18,281-325 3. Narita, K. (1958) Biochim. Bzbphys. Acta 28, 184-191 4. Harris, J. I. (1959) Biochem. J. 71,451-459 5. Brown, J. L., and Roberts, W. K. (1976) J. Biol. Chem. 2 5 1 , 1009-1014 6. Brown, J. L. (1979) J. Biol. Chem. 254,1447-1449 7. Wold, F. (1984) Trends Biochem. Sci. 9, 256-257 8. Jornvall, H. (1975) J. Theoret. Biol. 55, 1-12 9. Rubenstein, P., and Deuchler, J. (1979) J. Bid. Chem. 254, 11142-11147 10. Hershko, A., Heller, H., Eytan, E., Kaklij, G., and Rose, I. A. (1984) Proc. Nutl. Acad. Sci. U. S. A. 81, 7021-7025

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11. Bachmair, A., Finley, D., and Varshavsky, A. (1986) Science 2 3 4 , 179-186 12. Brot, N., Marcel, R., Cupp, L., and Weissbach, H. (1973) Arch. Biochem. Biophys. 155,475-477 13. Pestana, A., and Pitot, H.C. (1975) Biochemistry 1 4 , 1397-1403 14. Pestana, A., and Pitot, H.C. (1975) Biochemistry 1 4 , 1404-1412 15. Yamada, R., Kendall,R., and Bradshaw, R. A. (1987) First Symposium of the Protein Society, San Diego, CA, p. 34, Abstr. 625 16. O'Donohue, T. L. (1983) J. Biol. Chem. 2 5 8 , 2163-2167 17. Woodford, T. A., and Dixon, J. E. (1979) J. Biol. Chem. 254, 4993-4999 18. Pease, K. A., and Dixon, J. E. (1981) Arch. Biochem.Biophys. 2 1 2 , 177-185 19. Glembotski, C. C. (1982) J . Bid. Chen. 2 5 7 , 10501-10509 20. Chappell, M. C., O'Donohue, T. L., Millington, W. R., and Kempner, E. S.(1986) J. Bid. Chem. 2 6 1 , 1088-1091 21. Granger, M., Tesser, G. I., De Jong, W. W., and Blomendal, H. (1976) Proc. Natl. Acad. Sci. U. S. A. 7 3 , 3010-3014 22. Tsunasawa, S., Kamitani, K., and Narita, K. (1980) J . Biochem. (Tokyo)87,645-650 23. Kido, H., Vita, A,, Hannappel, E., and Horecker, B.L. (1981) Arch. Biochem. Biophys. 2 0 8 , 9 5 - 1 0 0 24. Lee, F.-J. S., Lin L.-W., and Smith, J. A. (1988) J. Biol. Chem. 263,14948-14955 25. Wong, W. W., Klickstein, L. B., Smith, J. A., Weis, J. H., and Fearon, D. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 77117715 26. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J . (1981) J . Biol. Chem. 2 5 6 , 7990-7997 27. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 28. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412 29. Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2 , 161-170 30. Gubler, U., and Hoffman, B. J . (1983) Gene (Amst.) 2 5 , 263-269 31. Aruffo, A., and Seed, B. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 8573-8577 32. Young, R. A,, and Davis, R. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,1194-1198 33. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 34. Lathe, R. (1985) J. Mol. Bid. 1 8 3 , 1-12

a cDNA Encoding Ne-Acetyltransferase 35. Matteucci, M. D., and Caruthers, M. H. (1981) J . Am. Chem. SOC. 1 0 3 , 3185-3191 36. Sinha, N.D., Biernat, J., McManus, J., and Koster, H. (1984) Nucleic Acids Res. 12, 4539-4544 37. Zoller, M., and Smith, M. (1985) DNA (NY) 3 , 479-488 38. Suggs, S. V., Hirose, T., Miyake, T., Kawashima, E. H., Johnson, M. J., Itakura, K., and Wallace, R. B. (1981) in Developmental Biology Using Purified Genes (Brown, D., ed) pp. 683-693, Academic Press, Orlando, FL 39. Henikoff, S. (1984) Gene (Amst.) 2 8 , 351-359 40. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448 41. Guo. L.-H. Yam, G. C. A., and Wu, R. (1983) Nucleic Acids Res. 1 i, 5521-5539 42. Lehrach, H., Dimond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16,4743-4751 43. Thomas. P. S. (1980) , . Proc. Natl. Acad. Sci. U. S. A. 77, 52015202 44. Southern, E. M. (1975) J . Mol. Biol. 9 8 , 503-517 45. Shore, D., Squire, M., and Nasmyth, K. A. (1984) EMBO J. 3 , 2817-2823 46. Itoh, N.,Slemmon, R., Hawke, D., Williamson, R., Morita, E., Itakura, K., Roberts, E., Shively, J. E., Crawford, G. D., and Salvaterra, P. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4081-4085 47. Shaw, W. V., Packman, L. C., Burleigh, B. D., Dell, A,, Morris, H. R., and Hartley, B. S. (1979) Nature 2 8 2 , 870-872 48. Yoshikawa, A,, Isono, S., Sheback, A,, and Isono, K. (1987) Mol. Gen. Genet. 209,481-488 49. Deguchi, T., Sakamoto, Y., Sasaki, Y., and Uyemura, K. (1988) J. Biol. Chem. 263, 7528-7533 50. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 12,387-395 51. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132 52. Chou, P. Y., and Fasman, G. D. (1977) in Peptides: Proceedings of the Fifth American Peptide Symposium (Goodman, M., and Meienhofer, J., eds) pp. 284-287, John Wiley & Sons, New York 53. Eberwine, J. H., Barchas, J. D., Hewlett, W. A., and Evans, C. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 1447-1453 54. Persson, B., Flinta, C., Heijne, G., and Jornvall, H. (1985) Eur. J . Biochem. 1 5 2 , 523-527 55. Augen, J., and Wold, F. (1986) Trends Biochem. Sci. 1 1 , 494497 56. Tsunasawa, S., Stewart, J . W., and Sherman, F. (1985) J. Biol. Chem. 260,5382-5391 57. Travis, G. H., Colavito-Shepanski, M., and Grunstein, M. (1984) J . Bid. Chem. 259,14406-14412

Molecular Cloning and Sequencing of a "x1

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