Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1995 Oct 16;14(20):5094–5099. doi: 10.1002/j.1460-2075.1995.tb00191.x

Maturase and endonuclease functions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA.

R M Henke 1, R A Butow 1, P S Perlman 1
PMCID: PMC394612  PMID: 7588637

Abstract

Intron 4 alpha (aI4 alpha) of the yeast mitochondrial COXI gene is a mobile group I intron that contains a reading frame encoding both the homing endonuclease I-SceII and a latent maturase capable of splicing both aI4 alpha and the fourth intron of the cytochrome b (COB) gene (bI4). The aI4 alpha reading frame is a member of a large gene family recognized by the presence of related dodecapeptide sequence motifs called P1 and P2. In this study, missense mutations of P1 and P2 were placed in mitochondrial DNA by biolistic transformation. The effects of the mutations on intron mobility, endonuclease I-SceII activity and maturase function were tested. The mutations of P1 strongly affected mobility and endonuclease I-SceII activity, but had little or no effect on maturase function; mutations of P2 affected splicing but not mobility or endonuclease I-SceII activity. Surprisingly, the conditional (temperature-sensitive) mutations at P1 and P2 block one or the other function of the protein but not both. This study indicates that the two functions depend on separate domains of the intron-encoded protein.

Full text

PDF
5096

Images in this article

Selected References

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

  1. Anziano P. Q., Hanson D. K., Mahler H. R., Perlman P. S. Functional domains in introns: trans-acting and cis-acting regions of intron 4 of the cob gene. Cell. 1982 Oct;30(3):925–932. doi: 10.1016/0092-8674(82)90297-5. [DOI] [PubMed] [Google Scholar]
  2. Bonitz S. G., Coruzzi G., Thalenfeld B. E., Tzagoloff A., Macino G. Assembly of the mitochondrial membrane system. Structure and nucleotide sequence of the gene coding for subunit 1 of yeast cytochrme oxidase. J Biol Chem. 1980 Dec 25;255(24):11927–11941. [PubMed] [Google Scholar]
  3. Cummings D. J., Michel F., McNally K. L. DNA sequence analysis of the 24.5 kilobase pair cytochrome oxidase subunit I mitochondrial gene from Podospora anserina: a gene with sixteen introns. Curr Genet. 1989 Dec;16(5-6):381–406. doi: 10.1007/BF00340719. [DOI] [PubMed] [Google Scholar]
  4. Cummings D. J., Michel F., McNally K. L. DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame. Curr Genet. 1989 Dec;16(5-6):407–418. doi: 10.1007/BF00340720. [DOI] [PubMed] [Google Scholar]
  5. De La Salle H., Jacq C., Slonimski P. P. Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase. Cell. 1982 Apr;28(4):721–732. doi: 10.1016/0092-8674(82)90051-4. [DOI] [PubMed] [Google Scholar]
  6. Delahodde A., Goguel V., Becam A. M., Creusot F., Perea J., Banroques J., Jacq C. Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria. Cell. 1989 Feb 10;56(3):431–441. doi: 10.1016/0092-8674(89)90246-8. [DOI] [PubMed] [Google Scholar]
  7. Dhawale S., Hanson D. K., Alexander N. J., Perlman P. S., Mahler H. R. Regulatory interactions between mitochondrial genes: interactions between two mosaic genes. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1778–1782. doi: 10.1073/pnas.78.3.1778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dujardin G., Jacq C., Slonimski P. P. Single base substitution in an intron of oxidase gene compensates splicing defects of the cytochrome b gene. Nature. 1982 Aug 12;298(5875):628–632. doi: 10.1038/298628a0. [DOI] [PubMed] [Google Scholar]
  9. Dürrenberger F., Rochaix J. D. Characterization of the cleavage site and the recognition sequence of the I-CreI DNA endonuclease encoded by the chloroplast ribosomal intron of Chlamydomonas reinhardtii. Mol Gen Genet. 1993 Jan;236(2-3):409–414. doi: 10.1007/BF00277141. [DOI] [PubMed] [Google Scholar]
  10. Gimble F. S., Stephens B. W. Substitutions in conserved dodecapeptide motifs that uncouple the DNA binding and DNA cleavage activities of PI-SceI endonuclease. J Biol Chem. 1995 Mar 17;270(11):5849–5856. doi: 10.1074/jbc.270.11.5849. [DOI] [PubMed] [Google Scholar]
  11. Hensgens L. A., Bonen L., de Haan M., van der Horst G., Grivell L. A. Two intron sequences in yeast mitochondrial COX1 gene: homology among URF-containing introns and strain-dependent variation in flanking exons. Cell. 1983 Feb;32(2):379–389. doi: 10.1016/0092-8674(83)90457-9. [DOI] [PubMed] [Google Scholar]
  12. Hodges R. A., Perler F. B., Noren C. J., Jack W. E. Protein splicing removes intervening sequences in an archaea DNA polymerase. Nucleic Acids Res. 1992 Dec 11;20(23):6153–6157. doi: 10.1093/nar/20.23.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hudspeth M. E., Shumard D. S., Tatti K. M., Grossman L. I. Rapid purification of yeast mitochondrial DNA in high yield. Biochim Biophys Acta. 1980 Dec 11;610(2):221–228. doi: 10.1016/0005-2787(80)90003-9. [DOI] [PubMed] [Google Scholar]
  14. Kostriken R., Strathern J. N., Klar A. J., Hicks J. B., Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell. 1983 Nov;35(1):167–174. doi: 10.1016/0092-8674(83)90219-2. [DOI] [PubMed] [Google Scholar]
  15. Labouesse M., Herbert C. J., Dujardin G., Slonimski P. P. Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene. EMBO J. 1987 Mar;6(3):713–721. doi: 10.1002/j.1460-2075.1987.tb04812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lamb M. R., Anziano P. Q., Glaus K. R., Hanson D. K., Klapper H. J., Perlman P. S., Mahler H. R. Functional domains in introns. RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b. J Biol Chem. 1983 Feb 10;258(3):1991–1999. [PubMed] [Google Scholar]
  17. 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]
  18. Lazowska J., Claisse M., Gargouri A., Kotylak Z., Spyridakis A., Slonimski P. P. Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships. J Mol Biol. 1989 Jan 20;205(2):275–289. doi: 10.1016/0022-2836(89)90341-0. [DOI] [PubMed] [Google Scholar]
  19. Lazowska J., Jacq C., Slonimski P. P. Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell. 1980 Nov;22(2 Pt 2):333–348. doi: 10.1016/0092-8674(80)90344-x. [DOI] [PubMed] [Google Scholar]
  20. Macreadie I. G., Scott R. M., Zinn A. R., Butow R. A. Transposition of an intron in yeast mitochondria requires a protein encoded by that intron. Cell. 1985 Jun;41(2):395–402. doi: 10.1016/s0092-8674(85)80012-x. [DOI] [PubMed] [Google Scholar]
  21. Moran J. V., Mecklenburg K. L., Sass P., Belcher S. M., Mahnke D., Lewin A., Perlman P. Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2. Nucleic Acids Res. 1994 Jun 11;22(11):2057–2064. doi: 10.1093/nar/22.11.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moran J. V., Wernette C. M., Mecklenburg K. L., Butow R. A., Perlman P. S. Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron. Nucleic Acids Res. 1992 Aug 11;20(15):4069–4076. doi: 10.1093/nar/20.15.4069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakagawa K., Morishima N., Shibata T. A maturase-like subunit of the sequence-specific endonuclease endo.SceI from yeast mitochondria. J Biol Chem. 1991 Jan 25;266(3):1977–1984. [PubMed] [Google Scholar]
  24. Shub D. A., Goodrich-Blair H. Protein introns: a new home for endonucleases. Cell. 1992 Oct 16;71(2):183–186. doi: 10.1016/0092-8674(92)90345-d. [DOI] [PubMed] [Google Scholar]
  25. Tzagoloff A., Akai A., Kurkulos M., Repetto B. Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli. J Biol Chem. 1988 Jan 15;263(2):850–856. [PubMed] [Google Scholar]
  26. Weiss-Brummer B., Rödel G., Schweyen R. J., Kaudewitz F. Expression of the split gene cob in yeast: evidence for a precursor of a "maturase" protein translated from intron 4 and preceding exons. Cell. 1982 Jun;29(2):527–536. doi: 10.1016/0092-8674(82)90169-6. [DOI] [PubMed] [Google Scholar]
  27. Wenzlau J. M., Saldanha R. J., Butow R. A., Perlman P. S. A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell. 1989 Feb 10;56(3):421–430. doi: 10.1016/0092-8674(89)90245-6. [DOI] [PubMed] [Google Scholar]
  28. Wernette C. M., Saldahna R., Perlman P. S., Butow R. A. Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae. J Biol Chem. 1990 Nov 5;265(31):18976–18982. [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES