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Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi


Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids1,2. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1)3, which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.

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Figure 1: Cryptic peroxisomal targeting signals in Gapdh and Pgk1 in U. maydis.
Figure 2: Dual targeting of GAPDH and PGK occurs in many fungi.
Figure 3: Peroxisomal localization of glycolytic enzymes is important for virulence and growth in U. maydis.


  1. 1

    De Duve, C. & Baudhuin, P. Peroxisomes (microbodies and related particles). Physiol. Rev. 46, 323–357 (1966)

    CAS  Article  Google Scholar 

  2. 2

    Poirier, Y., Antonenkov, V. D., Glumoff, T. & Hiltunen, J. K. Peroxisomal β-oxidation – a metabolic pathway with multiple functions. Biochim. Biophys. Acta 1763, 1413–1426 (2006)

    CAS  Article  Google Scholar 

  3. 3

    Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J. & Subramani, S. A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol. 108, 1657–1664 (1989)

    CAS  Article  Google Scholar 

  4. 4

    Gabaldon, T. Peroxisome diversity and evolution. Phil. Trans. R. Soc. Lond. B 365, 765–773 (2010)

    CAS  Article  Google Scholar 

  5. 5

    Graham, I. A. Seed storage oil mobilization. Annu. Rev. Plant Biol. 59, 115–142 (2008)

    CAS  Article  Google Scholar 

  6. 6

    Michels, P. A., Bringaud, F., Herman, M. & Hannaert, V. Metabolic functions of glycosomes in trypanosomatids. Biochim. Biophys. Acta 1763, 1463–1477 (2006)

    CAS  Article  Google Scholar 

  7. 7

    Jedd, G. Fungal evo-devo: organelles and multicellular complexity. Trends Cell Biol. 21, 12–19 (2011)

    CAS  Article  Google Scholar 

  8. 8

    Purdue, P. E. & Lazarow, P. B. Peroxisome biogenesis. Annu. Rev. Cell Dev. Biol. 17, 701–752 (2001)

    CAS  Article  Google Scholar 

  9. 9

    Hoepfner, D., Schildknegt, D., Braakman, I., Philippsen, P. & Tabak, H. F. Contribution of the endoplasmic reticulum to peroxisome formation. Cell 122, 85–95 (2005)

    CAS  Article  Google Scholar 

  10. 10

    Tabak, H. F. et al. Formation of peroxisomes: present and past. Biochim. Biophys. Acta 1763, 1647–1654 (2006)

    CAS  Article  Google Scholar 

  11. 11

    Girzalsky, W., Saffian, D. & Erdmann, R. Peroxisomal protein translocation. Biochim. Biophys. Acta 1803, 724–731 (2010)

    CAS  Article  Google Scholar 

  12. 12

    Brocard, C. & Hartig, A. Peroxisome targeting signal 1: is it really a simple tripeptide? Biochim. Biophys. Acta 1763, 1565–1573 (2006)

    CAS  Article  Google Scholar 

  13. 13

    Lazarow, P. B. The import receptor Pex7p and the PTS2 targeting sequence. Biochim. Biophys. Acta 1763, 1599–1604 (2006)

    CAS  Article  Google Scholar 

  14. 14

    Neuberger, G., Maurer-Stroh, S., Eisenhaber, B., Hartig, A. & Eisenhaber, F. Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J. Mol. Biol. 328, 581–592 (2003)

    CAS  Article  Google Scholar 

  15. 15

    Jones, J. M., Nau, K., Geraghty, M. T., Erdmann, R. & Gould, S. J. Identification of peroxisomal acyl-CoA thioesterases in yeast and humans. J. Biol. Chem. 274, 9216–9223 (1999)

    CAS  Article  Google Scholar 

  16. 16

    Baranov, P. V., Gesteland, R. F. & Atkins, J. F. Recoding: translational bifurcations in gene expression. Gene 286, 187–201 (2002)

    CAS  Article  Google Scholar 

  17. 17

    Gruber, A. R., Lorenz, R., Bernhart, S. H., Neubock, R. & Hofacker, I. L. The Vienna RNA websuite. Nucleic Acids Res. 36, W70–W74 (2008)

    CAS  Article  Google Scholar 

  18. 18

    Gurvitz, A. et al. The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19 . J. Biol. Chem. 272, 22140–22147 (1997)

    CAS  Article  Google Scholar 

  19. 19

    Managadze, D. et al. A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa . Proteomics 10, 3222–3234 (2010)

    CAS  Article  Google Scholar 

  20. 20

    Idnurm, A., Giles, S. S., Perfect, J. R. & Heitman, J. Peroxisome function regulates growth on glucose in the basidiomycete fungus Cryptococcus neoformans . Eukaryot. Cell 6, 60–72 (2007)

    CAS  Article  Google Scholar 

  21. 21

    Kiel, J. A., Veenhuis, M. & van der Klei, I. J. PEX genes in fungal genomes: common, rare or redundant. Traffic 7, 1291–1303 (2006)

    CAS  Article  Google Scholar 

  22. 22

    van Roermund, C. W., Elgersma, Y., Singh, N., Wanders, R. J. & Tabak, H. F. The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions. EMBO J. 14, 3480–3486 (1995)

    CAS  Article  Google Scholar 

  23. 23

    Visser, W. F., van Roermund, C. W., Ijlst, L., Waterham, H. R. & Wanders, R. J. Metabolite transport across the peroxisomal membrane. Biochem. J. 401, 365–375 (2007)

    CAS  Article  Google Scholar 

  24. 24

    Rottensteiner, H. & Theodoulou, F. L. The ins and outs of peroxisomes: co-ordination of membrane transport and peroxisomal metabolism. Biochim. Biophys. Acta 1763, 1527–1540 (2006)

    CAS  Article  Google Scholar 

  25. 25

    Bernhardt, K., Wilkinson, S., Weber, A. P. & Linka, N. A peroxisomal carrier delivers NAD and contributes to optimal fatty acid degradation during storage oil mobilization. Plant J. 69, 1–13 (2011)

    Article  Google Scholar 

  26. 26

    Kiel, J. A. et al. Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells. Funct. Integr. Genomics 9, 167–184 (2009)

    CAS  Article  Google Scholar 

  27. 27

    Strijbis, K., van den Burg, J., F, Visser, W., van den Berg, M. & Distel, B. Alternative splicing directs dual localization of Candida albicans 6-phosphogluconate dehydrogenase to cytosol and peroxisomes. FEMS Yeast Res. 12, 61–68 (2012)

    CAS  Article  Google Scholar 

  28. 28

    Kabran, P., Rossignol, T., Gaillardin, C., Nicaud, J. M. & Neuveglise, C. Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica . DNA Res. 10.1093/dnares/dss007 (24 February 2012)

  29. 29

    Teusink, B., Walsh, M. C., van Dam, K. & Westerhoff, H. V. The danger of metabolic pathways with turbo design. Trends Biochem. Sci. 23, 162–169 (1998)

    CAS  Article  Google Scholar 

  30. 30

    Haanstra, J. R. et al. Compartmentation prevents a lethal turbo-explosion of glycolysis in trypanosomes. Proc. Natl Acad. Sci. USA 105, 17718–17723 (2008)

    CAS  ADS  Article  Google Scholar 

  31. 31

    Sambrook, J. & Russell, D. Molecular Cloning: a Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2001)

    Google Scholar 

  32. 32

    Schulz, B. et al. The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif. Cell 60, 295–306 (1990)

    CAS  Article  Google Scholar 

  33. 33

    Loubradou, G., Brachmann, A., Feldbrügge, M. & Kahmann, R. A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis . Mol. Microbiol. 40, 719–730 (2001)

    CAS  Article  Google Scholar 

  34. 34

    Guthrie, C. & Fink, G. R. Guide to Yeast Genetics and Molecular and Cell Biology. (Academic, 2002)

    Google Scholar 

  35. 35

    Brachmann, A., König, J., Julius, C. & Feldbrügge, M. A reverse genetic approach for generating gene replacement mutants in Ustilago maydis . Mol. Genet. Genomics 272, 488 (2004)

    CAS  Article  Google Scholar 

  36. 36

    Schink, K. O. & Bölker, M. Coordination of cytokinesis and cell separation by endosomal targeting of a Cdc42-specific guanine nucleotide exchange factor in Ustilago maydis . Mol. Biol. Cell 20, 1081–1088 (2009)

    CAS  Article  Google Scholar 

  37. 37

    Spellig, T., Bottin, A. & Kahmann, R. Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis . Mol. Gen. Genet. 252, 503–509 (1996)

    CAS  PubMed  Google Scholar 

  38. 38

    Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004)

    CAS  Article  Google Scholar 

  39. 39

    Hoffman, C. S. & Winston, F. A ten minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation in Escherichia coli . Gene 57, 267–272 (1987)

    CAS  Article  Google Scholar 

  40. 40

    Kämper, J. et al. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis . Nature 444, 97–101 (2006)

    ADS  Article  Google Scholar 

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We are indebted to J. Heitman and W. K. Holloman for reading of the manuscript. We thank R. Fischer and G. Barth for providing strains and DNA. We acknowledge technical assistance by M. Piscator. We thank D. Lanver and T. Stehlik for discussions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG-GK1216). J.A. received a fellowship from the Marburg University Research Academy (MARA).

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M.B. and J.F. designed this study. J.F. and J.A. performed the experiments. All authors contributed to data analysis. M.B. and J.F. wrote the manuscript.

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Correspondence to Michael Bölker.

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The authors declare no competing financial interests.

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Freitag, J., Ast, J. & Bölker, M. Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi. Nature 485, 522–525 (2012).

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