Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Sep;12(9):4026–4037. doi: 10.1128/mcb.12.9.4026

DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae.

L C Mattheakis 1, W H Shen 1, R J Collier 1
PMCID: PMC360293  PMID: 1508200

Abstract

A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant. A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

Full text

PDF
4026

Images in this article

4029Image
on p.4029
4033Image
on p.4033
4033Image
on p.4033
4033Image
on p.4033
4034Image
on p.4034
4034Image
on p.4034

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Blanche F., Robin C., Couder M., Faucher D., Cauchois L., Cameron B., Crouzet J. Purification, characterization, and molecular cloning of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Methanobacterium ivanovii. J Bacteriol. 1991 Aug;173(15):4637–4645. doi: 10.1128/jb.173.15.4637-4645.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Carroll S. F., Barbieri J. T., Collier R. J. Diphtheria toxin: purification and properties. Methods Enzymol. 1988;165:68–76. doi: 10.1016/s0076-6879(88)65014-2. [DOI] [PubMed] [Google Scholar]
  5. Carroll S. F., Collier R. J. Diphtheria toxin: quantification and assay. Methods Enzymol. 1988;165:218–225. doi: 10.1016/s0076-6879(88)65034-8. [DOI] [PubMed] [Google Scholar]
  6. Chen J. Y., Bodley J. W. Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase. J Biol Chem. 1988 Aug 25;263(24):11692–11696. [PubMed] [Google Scholar]
  7. Chen J. Y., Bodley J. W., Livingston D. M. Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae. Mol Cell Biol. 1985 Dec;5(12):3357–3360. doi: 10.1128/mcb.5.12.3357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crouzet J., Cauchois L., Blanche F., Debussche L., Thibaut D., Rouyez M. C., Rigault S., Mayaux J. F., Cameron B. Nucleotide sequence of a Pseudomonas denitrificans 5.4-kilobase DNA fragment containing five cob genes and identification of structural genes encoding S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase and cobyrinic acid a,c-diamide synthase. J Bacteriol. 1990 Oct;172(10):5968–5979. doi: 10.1128/jb.172.10.5968-5979.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunlop P. C., Bodley J. W. Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae. J Biol Chem. 1983 Apr 25;258(8):4754–4758. [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Fendrick J. L., Iglewski W. J. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):554–557. doi: 10.1073/pnas.86.2.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Finley D., Bartel B., Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
  14. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  15. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  16. Ingrosso D., Fowler A. V., Bleibaum J., Clarke S. Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. J Biol Chem. 1989 Nov 25;264(33):20131–20139. [PubMed] [Google Scholar]
  17. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kohno K., Uchida T., Mekada E., Okada Y. Characterization of diphtheria-toxin-resistant mutants lacking receptor function or containing nonribosylatable elongation factor 2. Somat Cell Mol Genet. 1985 Sep;11(5):421–431. doi: 10.1007/BF01534836. [DOI] [PubMed] [Google Scholar]
  19. Kohno K., Uchida T., Ohkubo H., Nakanishi S., Nakanishi T., Fukui T., Ohtsuka E., Ikehara M., Okada Y. Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins. Proc Natl Acad Sci U S A. 1986 Jul;83(14):4978–4982. doi: 10.1073/pnas.83.14.4978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Lee H., Iglewski W. J. Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A. Proc Natl Acad Sci U S A. 1984 May;81(9):2703–2707. doi: 10.1073/pnas.81.9.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moehring J. M., Moehring T. J., Danley D. E. Posttranslational modification of elongation factor 2 in diphtheria-toxin-resistant mutants of CHO-K1 cells. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1010–1014. doi: 10.1073/pnas.77.2.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moehring J. M., Moehring T. J. The post-translational trimethylation of diphthamide studied in vitro. J Biol Chem. 1988 Mar 15;263(8):3840–3844. [PubMed] [Google Scholar]
  24. Moehring T. J., Danley D. E., Moehring J. M. In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin. Mol Cell Biol. 1984 Apr;4(4):642–650. doi: 10.1128/mcb.4.4.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Monia B. P., Haskell K. M., Ecker J. R., Ecker D. J., Crooke S. T. Chromosomal mapping of the ubiquitin gene family in Saccharomyces cerevisiae by pulsed field gel electrophoresis. Nucleic Acids Res. 1989 May 11;17(9):3611–3612. doi: 10.1093/nar/17.9.3611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Omura F., Kohno K., Uchida T. The histidine residue of codon 715 is essential for function of elongation factor 2. Eur J Biochem. 1989 Mar 1;180(1):1–8. doi: 10.1111/j.1432-1033.1989.tb14607.x. [DOI] [PubMed] [Google Scholar]
  27. Ozkaynak E., Finley D., Solomon M. J., Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. doi: 10.1002/j.1460-2075.1987.tb02384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pappenheimer A. M., Jr, Dunlop P. C., Adolph K. W., Bodley J. W. Occurrence of diphthamide in archaebacteria. J Bacteriol. 1983 Mar;153(3):1342–1347. doi: 10.1128/jb.153.3.1342-1347.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Peakman T., Crouzet J., Mayaux J. F., Busby S., Mohan S., Harborne N., Wootton J., Nicolson R., Cole J. Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-12 chromosome. Eur J Biochem. 1990 Jul 31;191(2):315–323. doi: 10.1111/j.1432-1033.1990.tb19125.x. [DOI] [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Perentesis J. P., Genbauffe F. S., Veldman S. A., Galeotti C. L., Livingston D. M., Bodley J. W., Murphy J. R. Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8386–8390. doi: 10.1073/pnas.85.22.8386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pfeifer K., Arcangioli B., Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. doi: 10.1016/0092-8674(87)90750-1. [DOI] [PubMed] [Google Scholar]
  33. Pósfai J., Bhagwat A. S., Pósfai G., Roberts R. J. Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res. 1989 Apr 11;17(7):2421–2435. doi: 10.1093/nar/17.7.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robin C., Blanche F., Cauchois L., Cameron B., Couder M., Crouzet J. Primary structure, expression in Escherichia coli, and properties of S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from Bacillus megaterium. J Bacteriol. 1991 Aug;173(15):4893–4896. doi: 10.1128/jb.173.15.4893-4896.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  40. Tweten R. K., Collier R. J. Molecular cloning and expression of gene fragments from corynebacteriophage beta encoding enzymatically active peptides of diphtheria toxin. J Bacteriol. 1983 Nov;156(2):680–685. doi: 10.1128/jb.156.2.680-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Van Ness B. G., Howard J. B., Bodley J. W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its hydrolysis products. J Biol Chem. 1980 Nov 25;255(22):10717–10720. [PubMed] [Google Scholar]
  42. Wilson B. A., Reich K. A., Weinstein B. R., Collier R. J. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. Biochemistry. 1990 Sep 18;29(37):8643–8651. doi: 10.1021/bi00489a021. [DOI] [PubMed] [Google Scholar]
  43. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  44. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES