Advertisement

Molecular Genetics and Genomics

, Volume 268, Issue 5, pp 637–644 | Cite as

Structure, organization and expression of the genes encoding mitochondrial cytochrome c 1 and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii

  • A. Atteia
  • R. van Lis
  • D. Wetterskog
  • E.-B. Gutiérrez-Cirlos
  • L. Ongay-Larios
  • L.-G. Franzén
  • D. González-Halphen
Original Paper

Abstract

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c 1 ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc 1 complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c 1 or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc 1 complex fail to assemble properly in the absence of cytochrome b.

Keywords

Expression of nucleus-encoded genes Introns Internal conserved sequences Mitochondrial targeting sequences Assembly of protein complexes 

Notes

Acknowledgments

The biochemical characterization of the dum-1 mutant was initiated in the Laboratory of Dr. J.-L Popot (IBPC, Paris). The authors wish to thank Drs. E. H. Harris (Duke University) and C. D. Silflow (University of Minnesota), who kindly provided us with DNA probes used in this work; Dr. H.-P. Braun (Institut fur Angewandte Genetik, Universität Hannover) and Dr. G. Schatz (Biozentrum der Universität Basel) for the gift of the antisera; Drs. J. Girard-Bascou (IBPC, Paris), E. H. Harris (Duke University) and R.F. Matagne (Laboratoire de Génétique des Microorganismes, Liège) for the gift of several probes and strains. We express our gratitude to T. Ballado (IFC, UNAM) for her valuable advice along the work, and to Dr. G. Dreyfus (IFC, UNAM) for the use of some of his laboratory equipment and chemicals. The technical skills of Guadalupe Códiz (Unidad de Biología Molecular, IFC, UNAM) in expert nucleotide sequencing are also acknowledged. This work was supported by grants from CONACyT, Mexico (27754 N); and from DGAPA, UNAM, Mexico (IN207201). RvL received a fellowship from DGEP (UNAM). LGF was supported by a grant from Carl Trygger's Foundation for Scientific Research

References

  1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1–15Google Scholar
  2. Atteia A (1994a) Identification of mitochondrial respiratory proteins from the green alga Chlamydomonas reinhardtii. C R Acad Sci III 317:11–19PubMedGoogle Scholar
  3. Atteia A (1994b) Contributions to the study of proteins of the mitochondrial respiratory chain in the green alga Chlamydomonas reinhardtii. Ph.D. dissertation, University of Paris VII, FranceGoogle Scholar
  4. Atteia A, Franzén L-G (1996) Identification, DNA sequence and deduced amino acid sequence of the mitochondrial Rieske iron-sulfur protein from the green alga Chlamydomonas reinhardtii. Implications from protein targeting and subunit interaction. Eur J Biochem 237:792–799PubMedGoogle Scholar
  5. Atteia A, de Vitry C, Pierre Y, Popot J-L (1992) Identification of mitochondrial proteins in membrane preparations from Chlamydomonas reinhardtii. J Biol Chem 267:226–234PubMedGoogle Scholar
  6. Atteia A, van Lis R, Ramírez J, González-Halphen D (2000) Polytomella spp. growth on ethanol. Extracellular pH affects the accumulation of mitochondrial cytochrome c 550. Eur J Biochem 267:2850–2858CrossRefPubMedGoogle Scholar
  7. Berry EA, Guergova-Kuras M, Huang L-S, Crofts AR (2000) Structure and function of cytochrome bc complexes. Annu Rev Biochem 69:1005–1075PubMedGoogle Scholar
  8. Braun HP, Emmermann M, Kruft V, Schmitz UK (1992) Cytochrome c 1 from potato: a protein with a presequence for targeting to the mitochondrial intermembrane space. Mol Gen Genet 231:217–225PubMedGoogle Scholar
  9. Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50:349–83PubMedGoogle Scholar
  10. Choquet Y, Wostrikoff K, Rimbault B, Zito F, Girard-Bascou J, Drapier D, Wollman F-A (2001) Assembly-controlled regulation of chloroplast gene translation. Biochem Soc Trans 29:421–426PubMedGoogle Scholar
  11. Chua N-H, Bennoun P (1975) Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center. Proc Natl Acad Sci USA 72:2175–2179PubMedGoogle Scholar
  12. Conner TW, Thompson MD, Silflow CD (1989) Structure of the three β-tubulin-encoding genes of the unicellular alga, Polytomella agilis. Gene 84:345–358PubMedGoogle Scholar
  13. Day A, Schirmer-Rahire M, Kuchka MR, Mayfield SP, Rochaix JD (1988) A transposon with an unusual arrangement of long terminal repeats in the green alga Chlamydomonas reinhardtii. EMBO J 7:1917–1927 PubMedGoogle Scholar
  14. Funke RP, Kovar JL, Logsdon Jr JM, Corrette-Bennet JC, Straus DR, Weeks DP (1999) Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reinhardtii and Volvox carteri: phylogenetic origins and recent insertion of introns. Mol Gen Genet 262:12–21PubMedGoogle Scholar
  15. Gutiérrez-Cirlos EB, Antaramian A, Vázquez-Acevedo M, Coria R, González-Halphen D (1994) A highly active ubiquinol-cytochrome c reductase ( bc 1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c 1. J Biol Chem 269:9147–9154PubMedGoogle Scholar
  16. Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San DiegoGoogle Scholar
  17. Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52:363–406PubMedGoogle Scholar
  18. Higgins DG, Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237–244PubMedGoogle Scholar
  19. Kaneko T, Kotani H, Nakamura Y, Sato S, Asamizu E, Miyajima N, Tabata S (1998) Structural analysis of Arabidopsis thaliana chromosome 5. V. Sequence features of the regions of 1,381,565 bp covered by twenty-one physically assigned P1 and TAC clones. DNA Res 5:131–145PubMedGoogle Scholar
  20. Liss M, Kirk DL, Beyser K, Fabry S (1997) Intron sequences provide a tool for high-resolution phylogenetic analysis of volvocine algae. Curr Genet 31:214–227CrossRefPubMedGoogle Scholar
  21. Matagne RF, Michel-Wolweretz MR, Munaut C, Duyckaerts C, Sluse F (1989) Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii. J Cell Biol. 108:1221–1226Google Scholar
  22. Michaelis G, Vahrenholz C, Pratje E (1990) Mitochondrial DNA of Chlamydomonas reinhardtii: the gene for apocytochrome b and the complete functional map of the 15.8-kb DNA. Mol Gen Genet 223:211–216PubMedGoogle Scholar
  23. Newman SM, Boynton JE, Gillham NW, Randolph-Anderson BL, Johnson AM, Harris EH (1990) Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events. Genetics 126:875–888PubMedGoogle Scholar
  24. Nishikimi M, Ohta S, Suzuki H, Tanaka T, Kikkawa F, Tanaka M, Kagawa Y, Ozawa T (1988) Nucleotide sequence of a cDNA encoding the precursor to human cytochrome c 1. Nucleic Acids Res16:3577Google Scholar
  25. Nurani G, Eriksson M, Knorpp C, Glaser E, Franzén L-G (1997) Homologous and heterologous protein import into mitochondria isolated from the green alga Chlamydomonas reinhardtii. Plant Mol Biol 35:973–980CrossRefPubMedGoogle Scholar
  26. Pérez-Martínez X, Funes S, Tolkunova E, Davidson E, King MP, González-Halphen D (2002) Structure of nuclear-localized cox3 genes in Chlamydomonas reinhardtii and in its colorless close relative Polytomella sp. Curr Genet 40:399–404PubMedGoogle Scholar
  27. Piccioni RG, Bennoun P, Chua N-H (1981) A nuclear mutant of Chlamydomonas reinhardtii defective in photosynthetic photophosphorylation. Eur J Biochem 117:93–102PubMedGoogle Scholar
  28. Randolph-Anderson BL, Boynton JE, Gillham NW, Harris EH, Johnson AM, Dorthu MP, Matagne RF (1993) Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet 236:235–244PubMedGoogle Scholar
  29. Randolph-Anderson BL, Sato R, Johnson AM, Harris EH, Hauser CR, Oeda K, Ishige F, Nishio S, Gillham NW, Boynton JE (1998) Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides. Plant Mol Biol 38:839–858CrossRefPubMedGoogle Scholar
  30. Remacle C, Duby F, Cardol P, Matagne RF (2001) Mutations inactivating mitochondrial genes in Chlamydomonas reinhardtii. Biochem Soc Trans 29:442–446PubMedGoogle Scholar
  31. Römisch J, Tropschug M, Sebald W, Weiss H (1987) The primary structure of cytochrome c 1 from Neurospora crassa. Eur J Biochem 164:111–115PubMedGoogle Scholar
  32. Sadler I, Suda K, Schatz G, Kaudewitz F, Haid A (1984) Sequencing of the nuclear gene for the yeast cytochrome c 1 precursor reveals an unusually complex amino-terminal presequence. EMBO J 3:2137–2143PubMedGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  34. Silflow C (1998) Organization of the nuclear genome. In: Rochaix JD, Goldschmidt-Clermont M, Merchant S (eds) The molecular biology of chloroplasts and mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, pp 25–40Google Scholar
  35. Suzuki H, Hosokawa Y, Nishikimi M, Ozawa T (1989) Structural organization of the human mitochondria cytochrome c 1 gene. J Biol Chem 264:1368–1374PubMedGoogle Scholar
  36. Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the detection of the peroxidase activity of P450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem75:168–176Google Scholar
  37. Tzagoloff A (1995) Ubiquinol-cytochrome- c oxidoreductase from Saccharomyces cerevisiae. Methods Enzymol 260:51–63PubMedGoogle Scholar
  38. Von Heijne G, Steppuhn J, Herrmann R (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180:535–545PubMedGoogle Scholar
  39. Wegener S, Schmitz UK (1993) The presequence of cytochrome c 1 from potato mitochondria is encoded on four exons. Curr Genet 24:256–259PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • A. Atteia
    • 1
  • R. van Lis
    • 1
  • D. Wetterskog
    • 2
  • E.-B. Gutiérrez-Cirlos
    • 3
  • L. Ongay-Larios
    • 4
  • L.-G. Franzén
    • 2
    • 5
  • D. González-Halphen
    • 1
  1. 1.Departamento de Genética Molecular, Instituto de Fisiología CelularUniversidad Nacional Autónoma de MéxicoMéxico D.F.Mexico
  2. 2.Department of Plant PhysiologyGöteborg UniversityGöteborgSweden
  3. 3.Department of BiochemistryDartmouth Medical SchoolHanoverUSA
  4. 4.Unidad de Biología MolecularInstituto de Fisiología CelularMéxico D.F.Mexico
  5. 5.School of Business and Engineering, NaturrumUniversity of HalmstadHalmstadSweden

Personalised recommendations