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Current Genetics

, Volume 26, Issue 5–6, pp 430–437 | Cite as

The function and specificity of the C-terminal tripeptide glyoxysomal targeting signal in Neurospora crassa

  • Priyal A. de Zoysa
  • Ian F. Connerton
Original Articles

Abstract

The function of the C-terminal tripeptide targeting signal responsible for microbody targeting in many eukaryotes has been investigated in the filamentous fugus Neurospora crassa. Using an in-vivo targeting assay that employs transformants carrying C-terminally-modified versions of the bacterial enzyme chloramphenicol acetyltransferase (CAT), it has been demonstrated that C-terminal tripeptide-dependent import occurs most efficiently in response to nutritional acetate-induction. Under these conditions Neurospora generates a specialized organelle, the glyoxysome, which carries the enzymes responsible for the glyoxylate cycle and can be distinguished from peroxisome-like microbodies that contain catalase. Moreover, several C-terminal peptides have been tested in this system to extend the tripeptide targeting consensus to A/C/G/S-H/K/Q/R-I/L/V. However, the tripeptide analogue, ARM, found at the C-terminus of the glyoxylate cycle enzyme isocitrate lyase in higher plants, does not apparently function here.

Key words

Neurospora crassa Glyoxysomes Membrane targeting Glyoxylate cycle 

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References

  1. Aitchison JD, Murray WW, Rachubinski RA (1991) The carboxyl terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes. J Biol Chem 166:23197–23203Google Scholar
  2. Atomi H, Veda M, Hikida M, Hishida T, Teninishi Y, Tanaka A (1990) Peroxisomal iso-citrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization. J Biochem 107:262–266Google Scholar
  3. Behari R, Baker A (1993) The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in-vitro system. J Biol Chem 268:7315–7322Google Scholar
  4. Bergmeyer HU (ed) (1974) Methods for enzyme analysis (3rd edn). Verlag Chemie, GermanyGoogle Scholar
  5. Collins ME, Briggs G, Sawyer C, Sheffield P, Connerton IF (1991) An inducible gene expression system for Neurospora crassa. Enzyme Microb Technol 13:400–403Google Scholar
  6. Connerton IF (1990) Premeiotic disruption of the Neurospora crassa malate synthase gene by native and divergent DNAs. Mol Gen Genet 223:319–323Google Scholar
  7. Connerton IF, Fincham JRS, Sandeman RA, Hynes MJ (1990) Comparison and cross-species expression of the Acetyl CoA Synthetase genes of the ascomycete fungi, Aspergillus nidulans and Neurospora crassa. Mol Microbiol 4:451–460Google Scholar
  8. Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEBS Lett 303:113–116Google Scholar
  9. Gainey LDS, Kolbe K, Connerton IF (1991) Molecular cloning and characterization of the acetate-inducible isocitrate lyase of Neurospora crassa: evidence for a second isozyme. Mol Gen Genet 229:253–260Google Scholar
  10. Gainey LDS, Connerton IF, Lewis EH, Turner G, Ballance DJ (1992) Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3). Curr Genet 21:43–47Google Scholar
  11. Gietl C (1990) Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. Proc Natl Acad Sci USA 87:5773–5777Google Scholar
  12. Gonzalez E (1991) The C-terminal domain of plant catalases: implications for a glyoxysomal targeting sequence. Eur J Biochem 199:211–215Google Scholar
  13. Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Mol Cell Biol 4:1044–1051Google Scholar
  14. Gould SJ, Keller G-A, Subramani S (1987) Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol 105:2923–2931Google Scholar
  15. Gould SJ, Keller G-A, Subramani S (1988) Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J Cell Biol 107:897–905Google Scholar
  16. Gould SJ, Keller G-A, Hosken N, Wilkinson J, Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108:1657–1664Google Scholar
  17. Gould SJ, Keller G-A, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S (1990) Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J 9:85–90Google Scholar
  18. Hanahan D (1985) In: DNA cloning. IRL Press, Oxford, UK, pp 109–135 (ed. Glover, D. pl.)Google Scholar
  19. Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–195Google Scholar
  20. Johanson RA, Hill JM, McFadden BA (1974) Isocitrate lyase from Neurospora crassa. II. Composition, quaternary structure, C-terminus, and active-site modification. Biochim Biophys Acta 364:341–352Google Scholar
  21. Keller G-A, Krisans S, Gould SJ, Sommer JM, Wang CC, Schliebs W, Kunau W-H, Brody S, Subramani S (1991) Evolutionary conservation of microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J Cell Biol 114:893–904Google Scholar
  22. Kionka C, Kunau W-H (1985) Inducible β-oxidation pathway in Neurospora crassa. J Bacteriol 161:153–157Google Scholar
  23. Kornberg HL (1966) The role and control of the glyoxylate cycle in Escherichia coli. Biochem J 99:1–11Google Scholar
  24. Matsuoka M, McFadden BA (1988) Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in E. coli. J Bacteriol 170:4528–4536Google Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  26. Sandeman RA, Hynes MJ, Fincham JRS, Connerton IF (1991) Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa. Mol Gen Genet 228:445–452Google Scholar
  27. Schwitzguebel JP, Moller IM, Palmer JM (1981) Changes in density of mitochondria and glyoxysomes from Neurospora crassa: a reevaluation utilizing silica sol gradient centrifugation. J Gen Microbiol 126:289–295Google Scholar
  28. Small GM, Szabo LJ, Lazarow PB (1988) Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. EMBO J 7:1167–1173Google Scholar
  29. Stevens JN, Metzenberg RL (1982) An easy method for preparing Neurospora DNA. Neurospora Newslett 29:27–28Google Scholar
  30. Swinkels BW, Gould SJ, Subramani S (1992) Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett 305:133–136Google Scholar
  31. Thomas GH, Connerton IF, Fincham JRS (1988) Molecular cloning, identification and transcriptional analysis of genes involved in acetate utilization. Mol Microbiol 2:599–606Google Scholar
  32. Turley RB, Choe SM, Trelease RN (1990) Characterization of a cDNA clone encoding the complete amino-acid sequence of cotton isocitrate lyase. Biochim Biophys Acta 1049:223–226Google Scholar
  33. Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microb Genet Bull 13:42–43Google Scholar
  34. Vollmer SJ, Yanofsky C (1986) Efficiency cloning of genes of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873Google Scholar
  35. Wanner G, Theimer RR (1982) Two types of microbodies in Neurospora crassa. Ann NY Acad Sci 286:269–284Google Scholar
  36. Zimmerman R, Neupert W (1980) Biogenesis of glyoxysomes: synthesis and intracellular transfer of isocitrate lyase. Eur J Biochem 112:225–233Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Priyal A. de Zoysa
    • 1
  • Ian F. Connerton
    • 1
  1. 1.Department of MicrobiologyUniversity of ReadingReadingUK

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