Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Dec;12(12):5593–5599. doi: 10.1128/mcb.12.12.5593

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

K K Singh 1, G M Small 1, A S Lewin 1
PMCID: PMC360498  PMID: 1448089

Abstract

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.

Full text

PDF
5593

Images in this article

5596Image
on p.5596
5597Image
on p.5597

Selected References

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

  1. Baudhuin P., Beaufay H., Rahman-Li Y., Sellinger O. Z., Wattiaux R., Jacques P., De Duve C. Tissue fractionation studies. 17. Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue. Biochem J. 1964 Jul;92(1):179–184. doi: 10.1042/bj0920179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Clayton D. A. Nuclear gadgets in mitochondrial DNA replication and transcription. Trends Biochem Sci. 1991 Mar;16(3):107–111. doi: 10.1016/0968-0004(91)90043-u. [DOI] [PubMed] [Google Scholar]
  4. Cohen G., Rapatz W., Ruis H. Sequence of the Saccharomyces cerevisiae CTA1 gene and amino acid sequence of catalase A derived from it. Eur J Biochem. 1988 Sep 1;176(1):159–163. doi: 10.1111/j.1432-1033.1988.tb14263.x. [DOI] [PubMed] [Google Scholar]
  5. Danpure C. J., Cooper P. J., Wise P. J., Jennings P. R. An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria. J Cell Biol. 1989 Apr;108(4):1345–1352. doi: 10.1083/jcb.108.4.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dmochowska A., Dignard D., Maleszka R., Thomas D. Y. Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme A oxidase. Gene. 1990 Apr 16;88(2):247–252. doi: 10.1016/0378-1119(90)90038-s. [DOI] [PubMed] [Google Scholar]
  7. Erdmann R., Wiebel F. F., Flessau A., Rytka J., Beyer A., Fröhlich K. U., Kunau W. H. PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases. Cell. 1991 Feb 8;64(3):499–510. doi: 10.1016/0092-8674(91)90234-p. [DOI] [PubMed] [Google Scholar]
  8. Gasser S. M., Daum G., Schatz G. Import of proteins into mitochondria. Energy-dependent uptake of precursors by isolated mitochondria. J Biol Chem. 1982 Nov 10;257(21):13034–13041. [PubMed] [Google Scholar]
  9. Gould S. J., Keller G. A., Schneider M., Howell S. H., Garrard L. J., Goodman J. M., Distel B., Tabak H., Subramani S. Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J. 1990 Jan;9(1):85–90. doi: 10.1002/j.1460-2075.1990.tb08083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haas L. O., Cregg J. M., Gleeson M. A. Development of an integrative DNA transformation system for the yeast Candida tropicalis. J Bacteriol. 1990 Aug;172(8):4571–4577. doi: 10.1128/jb.172.8.4571-4577.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hijikata M., Ishii N., Kagamiyama H., Osumi T., Hashimoto T. Structural analysis of cDNA for rat peroxisomal 3-ketoacyl-CoA thiolase. J Biol Chem. 1987 Jun 15;262(17):8151–8158. [PubMed] [Google Scholar]
  12. Hiltunen J. K., Wenzel B., Beyer A., Erdmann R., Fosså A., Kunau W. H. Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product. J Biol Chem. 1992 Apr 5;267(10):6646–6653. [PubMed] [Google Scholar]
  13. Jones D. H., Howard B. H. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. Biotechniques. 1991 Jan;10(1):62–66. [PubMed] [Google Scholar]
  14. Keller G. A., Gould S., Deluca M., Subramani S. Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci U S A. 1987 May;84(10):3264–3268. doi: 10.1073/pnas.84.10.3264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kispal G., Evans C. T., Malloy C., Srere P. A. Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme. J Biol Chem. 1989 Jul 5;264(19):11204–11210. [PubMed] [Google Scholar]
  16. Kispal G., Rosenkrantz M., Guarente L., Srere P. A. Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases. J Biol Chem. 1988 Aug 15;263(23):11145–11149. [PubMed] [Google Scholar]
  17. Lazarow P. B. Peroxisome biogenesis. Curr Opin Cell Biol. 1989 Aug;1(4):630–634. doi: 10.1016/0955-0674(89)90026-4. [DOI] [PubMed] [Google Scholar]
  18. Lewin A. S., Hines V., Small G. M. Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal. Mol Cell Biol. 1990 Apr;10(4):1399–1405. doi: 10.1128/mcb.10.4.1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li J. M., Hopper A. K., Martin N. C. N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae. J Cell Biol. 1989 Oct;109(4 Pt 1):1411–1419. doi: 10.1083/jcb.109.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liao X. S., Small W. C., Srere P. A., Butow R. A. Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):38–46. doi: 10.1128/mcb.11.1.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McCammon M. T., Veenhuis M., Trapp S. B., Goodman J. M. Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae. J Bacteriol. 1990 Oct;172(10):5816–5827. doi: 10.1128/jb.172.10.5816-5827.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  23. Purdue P. E., Allsop J., Isaya G., Rosenberg L. E., Danpure C. J. Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10900–10904. doi: 10.1073/pnas.88.23.10900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosenkrantz M., Alam T., Kim K. S., Clark B. J., Srere P. A., Guarente L. P. Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes. Mol Cell Biol. 1986 Dec;6(12):4509–4515. doi: 10.1128/mcb.6.12.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Small G. M., Szabo L. J., Lazarow P. B. Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. EMBO J. 1988 Apr;7(4):1167–1173. doi: 10.1002/j.1460-2075.1988.tb02927.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smeekens S., Weisbeek P., Robinson C. Protein transport into and within chloroplasts. Trends Biochem Sci. 1990 Feb;15(2):73–76. doi: 10.1016/0968-0004(90)90180-j. [DOI] [PubMed] [Google Scholar]
  28. Swinkels B. W., Gould S. J., Bodnar A. G., Rachubinski R. A., Subramani S. A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J. 1991 Nov;10(11):3255–3262. doi: 10.1002/j.1460-2075.1991.tb04889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Veenhuis M., Mateblowski M., Kunau W. H., Harder W. Proliferation of microbodies in Saccharomyces cerevisiae. Yeast. 1987 Jun;3(2):77–84. doi: 10.1002/yea.320030204. [DOI] [PubMed] [Google Scholar]
  31. Yoo H. S., Cooper T. G. The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene's expression. Mol Cell Biol. 1989 Aug;9(8):3231–3243. doi: 10.1128/mcb.9.8.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES