Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1997 Sep;147(1):73–85. doi: 10.1093/genetics/147.1.73

Probing Novel Elements for Protein Splicing in the Yeast Vma1 Protozyme: A Study of Replacement Mutagenesis and Intragenic Suppression

S Nogami 1, Y Satow 1, Y Ohya 1, Y Anraku 1
PMCID: PMC1208124  PMID: 9286669

Abstract

Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions. This unique autocatalytic reaction was first discovered in the yeast Vma1p protozyme where the 50-kD site-specific endonuclease (VDE) is excised from the 120-kD precursor containing the N-and C-terminal regions of the catalytic subunit of the vacuolar H(+)-ATPase. In this work, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the Vma1 protozyme and found that these site-specific random mutations interfere with normal protein splicing to different extents. Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal splicing junction genetically interacts with three hydrophobic residues preceding the N-terminal splicing junction. This is the first evidence showing that the N-terminal portion of the V-ATPase subunit is involved in protein splicing. Our genetic evidence is consistent with a structural model that correctly aligns two parallel β-strands ascribed to the triplets. This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions.

Full Text

The Full Text of this article is available as a PDF (4.3 MB).

Selected References

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

  1. Abrahams J. P., Leslie A. G., Lutter R., Walker J. E. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 1994 Aug 25;370(6491):621–628. doi: 10.1038/370621a0. [DOI] [PubMed] [Google Scholar]
  2. Anraku Y., Hirata R. Protozyme: emerging evidence in nature. J Biochem. 1994 Feb;115(2):175–178. doi: 10.1093/oxfordjournals.jbchem.a124313. [DOI] [PubMed] [Google Scholar]
  3. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  4. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  5. Chong S., Shao Y., Paulus H., Benner J., Perler F. B., Xu M. Q. Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem. 1996 Sep 6;271(36):22159–22168. doi: 10.1074/jbc.271.36.22159. [DOI] [PubMed] [Google Scholar]
  6. Cooper A. A., Chen Y. J., Lindorfer M. A., Stevens T. H. Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision. EMBO J. 1993 Jun;12(6):2575–2583. doi: 10.1002/j.1460-2075.1993.tb05913.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis E. O., Jenner P. J., Brooks P. C., Colston M. J., Sedgwick S. G. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell. 1992 Oct 16;71(2):201–210. doi: 10.1016/0092-8674(92)90349-h. [DOI] [PubMed] [Google Scholar]
  8. Davis E. O., Thangaraj H. S., Brooks P. C., Colston M. J. Evidence of selection for protein introns in the recAs of pathogenic mycobacteria. EMBO J. 1994 Feb 1;13(3):699–703. doi: 10.1002/j.1460-2075.1994.tb06309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geourjon C., Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci. 1995 Dec;11(6):681–684. doi: 10.1093/bioinformatics/11.6.681. [DOI] [PubMed] [Google Scholar]
  10. Gimble F. S., Thorner J. Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae. Nature. 1992 May 28;357(6376):301–306. doi: 10.1038/357301a0. [DOI] [PubMed] [Google Scholar]
  11. Gu H. H., Xu J., Gallagher M., Dean G. E. Peptide splicing in the vacuolar ATPase subunit A from Candida tropicalis. J Biol Chem. 1993 Apr 5;268(10):7372–7381. [PubMed] [Google Scholar]
  12. Hirata R., Anraku Y. Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing. Biochem Biophys Res Commun. 1992 Oct 15;188(1):40–47. doi: 10.1016/0006-291x(92)92347-z. [DOI] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  15. Kane P. M., Yamashiro C. T., Wolczyk D. F., Neff N., Goebl M., Stevens T. H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science. 1990 Nov 2;250(4981):651–657. doi: 10.1126/science.2146742. [DOI] [PubMed] [Google Scholar]
  16. Kawasaki M., Makino S., Matsuzawa H., Satow Y., Ohya Y., Anraku Y. Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme. Biochem Biophys Res Commun. 1996 May 24;222(3):827–832. doi: 10.1006/bbrc.1996.0826. [DOI] [PubMed] [Google Scholar]
  17. Kawasaki M., Nogami S., Satow Y., Ohya Y., Anraku Y. Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme. A random mutagenesis study of the entire Vma1-derived endonuclease sequence. J Biol Chem. 1997 Jun 20;272(25):15668–15674. doi: 10.1074/jbc.272.25.15668. [DOI] [PubMed] [Google Scholar]
  18. Lambowitz A. M., Belfort M. Introns as mobile genetic elements. Annu Rev Biochem. 1993;62:587–622. doi: 10.1146/annurev.bi.62.070193.003103. [DOI] [PubMed] [Google Scholar]
  19. Lifson S., Sander C. Antiparallel and parallel beta-strands differ in amino acid residue preferences. Nature. 1979 Nov 1;282(5734):109–111. doi: 10.1038/282109a0. [DOI] [PubMed] [Google Scholar]
  20. Lifson S., Sander C. Specific recognition in the tertiary structure of beta-sheets of proteins. J Mol Biol. 1980 Jun 5;139(4):627–639. doi: 10.1016/0022-2836(80)90052-2. [DOI] [PubMed] [Google Scholar]
  21. Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
  22. Ohya Y., Umemoto N., Tanida I., Ohta A., Iida H., Anraku Y. Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity. J Biol Chem. 1991 Jul 25;266(21):13971–13977. [PubMed] [Google Scholar]
  23. Perler F. B., Comb D. G., Jack W. E., Moran L. S., Qiang B., Kucera R. B., Benner J., Slatko B. E., Nwankwo D. O., Hempstead S. K. Intervening sequences in an Archaea DNA polymerase gene. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5577–5581. doi: 10.1073/pnas.89.12.5577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Perler F. B., Davis E. O., Dean G. E., Gimble F. S., Jack W. E., Neff N., Noren C. J., Thorner J., Belfort M. Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. Nucleic Acids Res. 1994 Apr 11;22(7):1125–1127. doi: 10.1093/nar/22.7.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perler F. B., Olsen G. J., Adam E. Compilation and analysis of intein sequences. Nucleic Acids Res. 1997 Mar 15;25(6):1087–1093. doi: 10.1093/nar/25.6.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pietrokovski S. A new intein in cyanobacteria and its significance for the spread of inteins. Trends Genet. 1996 Aug;12(8):287–288. doi: 10.1016/0168-9525(96)20005-8. [DOI] [PubMed] [Google Scholar]
  27. Pietrokovski S. Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Sci. 1994 Dec;3(12):2340–2350. doi: 10.1002/pro.5560031218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shih C. K., Wagner R., Feinstein S., Kanik-Ennulat C., Neff N. A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. Mol Cell Biol. 1988 Aug;8(8):3094–3103. doi: 10.1128/mcb.8.8.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Story R. M., Weber I. T., Steitz T. A. The structure of the E. coli recA protein monomer and polymer. Nature. 1992 Jan 23;355(6358):318–325. doi: 10.1038/355318a0. [DOI] [PubMed] [Google Scholar]
  31. Xu M. Q., Comb D. G., Paulus H., Noren C. J., Shao Y., Perler F. B. Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. EMBO J. 1994 Dec 1;13(23):5517–5522. doi: 10.1002/j.1460-2075.1994.tb06888.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Xu M. Q., Southworth M. W., Mersha F. B., Hornstra L. J., Perler F. B. In vitro protein splicing of purified precursor and the identification of a branched intermediate. Cell. 1993 Dec 31;75(7):1371–1377. doi: 10.1016/0092-8674(93)90623-x. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES