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. 1986 Jul 15;237(2):421–426. doi: 10.1042/bj2370421

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants.

G J Threadgill, R C Conrad, L M Changchien, M Cannon, G R Craven
PMCID: PMC1147002  PMID: 3541900

Abstract

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.

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Selected References

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

  1. Carter C. J., Cannon M., Jiménez A. A trichodermin-resistant mutant of Saccharomyces cerevisiae with an abnormal distribution of native ribosomal subunits. Eur J Biochem. 1980;107(1):173–183. doi: 10.1111/j.1432-1033.1980.tb04638.x. [DOI] [PubMed] [Google Scholar]
  2. Carter C. J., Cannon M. Maturation of ribosomal precursor RNA in Saccharomyces cerevisiae. A mutant with a defect in both the transport and terminal processing of the 20 S species. J Mol Biol. 1980 Oct 25;143(2):179–199. doi: 10.1016/0022-2836(80)90197-7. [DOI] [PubMed] [Google Scholar]
  3. Cooper D., Banthorpe D. V., Wilkie D. Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. J Mol Biol. 1967 Jun 14;26(2):347–350. doi: 10.1016/0022-2836(67)90302-6. [DOI] [PubMed] [Google Scholar]
  4. Dalrymple P. N., Gupta S., Regnier F., Houston L. L. Separation of eucaryotic ribosomal proteins by high-performance liquid chromatography using a new cation-exchange resin and LiCl elution in 4 M urea. Biochim Biophys Acta. 1983 Jan 25;755(2):157–162. doi: 10.1016/0304-4165(83)90198-8. [DOI] [PubMed] [Google Scholar]
  5. Ferris R. J., Cowgill C. A., Traut R. R. Separation of ribosomal proteins from Escherichia coli and rabbit reticulocytes using reverse-phase high-performance liquid chromatography. Biochemistry. 1984 Jul 17;23(15):3434–3442. doi: 10.1021/bi00310a009. [DOI] [PubMed] [Google Scholar]
  6. Fried H. M., Warner J. R. Cloning of yeast gene for trichodermin resistance and ribosomal protein L3. Proc Natl Acad Sci U S A. 1981 Jan;78(1):238–242. doi: 10.1073/pnas.78.1.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grant P. G., Schindler D., Davies J. E. Mapping of trichodermin resistance in Saccharomyces cerevisiae: a genetic locus for a component of the 60S ribsomal subunit. Genetics. 1976 Aug;83(4):667–673. doi: 10.1093/genetics/83.4.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grant P., Sánchez L., Jiménez A. Cryptopleurine resistance: genetic locus for a 40S ribosomal component in Saccharomyces cerevisiae. J Bacteriol. 1974 Dec;120(3):1308–1314. doi: 10.1128/jb.120.3.1308-1314.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gritz L. R., Mitlin J. A., Cannon M., Littlewood B., Carter C. J., Davies J. E. Ribosome structure, maturation of ribosomal RNA and drug sensitivity in temperature-sensitive mutants of Saccharomyces cerevisiae. Mol Gen Genet. 1982;188(3):384–391. doi: 10.1007/BF00330038. [DOI] [PubMed] [Google Scholar]
  10. Hardy S. J., Kurland C. G., Voynow P., Mora G. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry. 1969 Jul;8(7):2897–2905. doi: 10.1021/bi00835a031. [DOI] [PubMed] [Google Scholar]
  11. Jimenez A., Sanchez L., Vazquez D. Simultaneous ribosomal resistance to trichodermin and anisomycin in Saccharomyces cerevisiae mutants. Biochim Biophys Acta. 1975 Apr 2;383(4):427–434. doi: 10.1016/0005-2787(75)90312-3. [DOI] [PubMed] [Google Scholar]
  12. Kamp R. M., Bosserhoff A., Kamp D., Wittmann-Liebold B. Application of high-performance liquid chromatographic techniques to the separation of ribosomal proteins of different organisms. J Chromatogr. 1984 Dec 28;317:181–192. doi: 10.1016/s0021-9673(01)91658-9. [DOI] [PubMed] [Google Scholar]
  13. Kerlavage A. R., Weitzmann C. J., Hasan T., Cooperman B. S. Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins. Characteristics of the separation of a complex protein mixture. J Chromatogr. 1983 Aug 26;266:225–237. doi: 10.1016/s0021-9673(01)90896-9. [DOI] [PubMed] [Google Scholar]
  14. Käufer N. F., Fried H. M., Schwindinger W. F., Jasin M., Warner J. R. Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 1983 May 25;11(10):3123–3135. doi: 10.1093/nar/11.10.3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martini O. H., Gould H. J. Enumeration of rabbit reticulocyte ribosomal proteins. J Mol Biol. 1971 Dec 14;62(2):403–405. doi: 10.1016/0022-2836(71)90435-9. [DOI] [PubMed] [Google Scholar]
  16. Mitlin J. A., Cannon M. Defective processing of ribosomal precursor RNA in Saccharomyces cerevisiae. Biochem J. 1984 Jun 1;220(2):461–467. doi: 10.1042/bj2200461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. REISFELD R. A., LEWIS U. J., WILLIAMS D. E. Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature. 1962 Jul 21;195:281–283. doi: 10.1038/195281a0. [DOI] [PubMed] [Google Scholar]
  18. Schindler D., Grant P., Davies J. Trichodermin resistance--mutation affecting eukaryotic ribosomes. Nature. 1974 Apr 5;248(448):535–536. doi: 10.1038/248535a0. [DOI] [PubMed] [Google Scholar]
  19. Schultz L. D., Friesen J. D. Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae. J Bacteriol. 1983 Jul;155(1):8–14. doi: 10.1128/jb.155.1.8-14.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Skogerson L., McLaughlin C., Wakatama E. Modification of ribosomes in cryptopleurine-resistant mutants of yeast. J Bacteriol. 1973 Nov;116(2):818–822. doi: 10.1128/jb.116.2.818-822.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stöcklein W., Piepersberg W., Böck A. Amino acid replacements in ribosomal protein YL24 of Saccharomyces cerevisiae causing resistance to cycloheximide. FEBS Lett. 1981 Dec 28;136(2):265–268. doi: 10.1016/0014-5793(81)80632-1. [DOI] [PubMed] [Google Scholar]

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