Skip to main content
SpringerLink
Log in

Characterization of peroxisome-deficient mutants of Hansenula polymorpha

  • Original Paper
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-). In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes. Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant per strains and wild-type H. polymorpha were Mut- and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Boeke JD, Trueheart J, Natsoulis G, Fink GR (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154:164–175

    Google Scholar 

  • Borst P (1989) Peroxisome biogenesis revisited. Biochim Biophys Acta 1008:1–13

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Brul S, Westerveld A, Strijland A, Wanders RJA., Schram A, Heymans W, Schutgens HSA, van den Bosch RBH, Tager JM (1988) Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions: a study using complementation analysis. J Clin Invest 81:1710–1715

    Google Scholar 

  • Cregg JM (1987) Genetics of methylotrophic yeasts. In: Duine JA, van Verseveld HW (eds) Proceedings of the fifth international symposium on microbial growth on C1 compounds. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp 158–167

    Google Scholar 

  • Cregg JM, van der Klei IJ, Sulter GJ, Veenhuis M, Harder W (1990) Peroxisome-deficient mutants of Hansenula polymorpha. Yeast 6:87–97

    Google Scholar 

  • Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4:293–303

    Google Scholar 

  • Didion T, Roggenkamp R (1990) Deficiency of peroxisome assembly in a mutant of the methylotrophic yeast Hansenula polymorpha. Curr Genet 17:113–117

    Google Scholar 

  • Elgersma Y, van den Berg M, Tabak HF, Distel B (1993) An efficient positive selection procedure for the isolation of peroxisomalimport and peroxisome-assembly mutants of Saccharomyces cerevisiae. Genetics 135:731–740

    Google Scholar 

  • Erdmann R, Veenhuis M, Mertens D, Kunau W-H (1989) Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 86:2432–2436

    Google Scholar 

  • Faber KN, Swaving GJ, Faber F, AB G, Harder W, Veenhuis M, Haima P (1992) Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. J Gen Microbiol 138:2405–2416

    Google Scholar 

  • Fujiki Y, Rachubinski RA, Lazarow PB (1984) Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes. Proc Natl Acad Sci USA 81:7127–7131

    Google Scholar 

  • Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4:293–303

    Google Scholar 

  • Goldfischer S, Moor CL, Johnson AB, Spiro AJ, Valsamis MP, Wisniewski HK, Ritch RH, Norton WT, Rapin I, Garner LM (1973). Peroxisomal and mitochondrial defects in cerebrohepatorenal syndrome. Science 182:62–64

    Google Scholar 

  • Gould SJ, Keller G-A, Subramani S (1987) Identification of peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol 105:2923–2931

    Google Scholar 

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

    Google Scholar 

  • 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 9:85–90

    Google Scholar 

  • Gould SJ, McCollum D, Spong AP, Heyman JA, Subramani S (1992) Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly. Yeast 8:613–628

    Google Scholar 

  • Hoop MJ de, AB G (1992) Import of proteins into peroxisomes and other microbodies. Biochem J 286:657–669

    Google Scholar 

  • Imanaka T, Small GM, Lazarow PB (1987) Translocation of acyl-CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential. J Cell Biol 105:2915–2922

    Google Scholar 

  • Keller G-A, Krisans S, Gould SJ, Sommer JM, Wang CC, Schliebs W, Kunau W, Brody S, Subramani S (1991) Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes. J Cell Biol 114:893–904

    Google Scholar 

  • Klei IJ van der, Bystrykh LV, Harder W (1990) Alcohol oxidase from Hansenula polymorpha CBS 4732. Methods Enzymol 188:420–422

    Google Scholar 

  • Klei IJ van der, Harder W, Veenhuis M (1991a) Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study. Arch Microbiol 156:15–23

    Google Scholar 

  • Klei IJ van der, Sulter GJ, Harder W, Veenhuis M (1991b) Expression, assembly and crystallization of alcohol oxidase in a peroxisome-deficient mutant of Hansenula polymorpha: properties of the protein and architecture of the crystals, Yeast 7:15–24

    Google Scholar 

  • Kunau W-H, Hartig A (1992) Peroxisome biogenesis in Saccharomyces cerevisiae. Antyan Leeuwenhoek J Microbiol Serol 62:63–78

    Google Scholar 

  • Lazarow PB (1993) Genetic approaches to studying peroxisome biogenesis. Trends Cell Biol 3:89–93

    Google Scholar 

  • Lazarow PB, Fujiki Y (1985) Biogenesis of peroxisomes. Annu Rev Cell Biol 1:489–530

    Google Scholar 

  • Lazarow PB, Moser HW (1989) Disorders of peroxisome biogenesis. In: Scriver CR, Beaudat AL, Sly WS, Valle D (eds) The metabolic basis of inherited diseases. McGraw-Hill, New York, pp 1479–1509

    Google Scholar 

  • Leij I van der, van den Berg M, Boot R, Franse M, Distel B, Tabak HF (1992) Isolation of peroxisome-assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure. J Cell Biol 119:153–162

    Google Scholar 

  • Leij I van der, Franse MM, Elgersma Y, Distel B, Tabak HF (1993) PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 90:11782–11786

    Google Scholar 

  • Liu H, Tan X, Veenhuis M, McCollum D, Cregg JM (1992) An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J Bacteriol 174:4943–4951

    Google Scholar 

  • Marzioch M, Erdmann R, Veenhuis M, Kunau W-H (1994) PAS7 encodes a novel yeast member of the WD-40 protein family essential for import of 3-oxoacyl-CoA thiolase, a PTS2-containing protein, into peroxisomes. EMBO J 13:4908–4918

    Google Scholar 

  • McCollum D, Monosov E, Subramani S (1993) The pas8 mutant of Pichia pastoris exhitits the peroxisomal protein import deficiencies of Zellweger syndrome cells-the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family. J Cell Biol 121:761–774

    Google Scholar 

  • Nuttley WM, Brade AM, Gaillardin C, Eitzen GA, Glover JR, Aitchison, JD, Rachubinski RA (1993) Rapid identification and characterization of peroxisome-assembly mutants in Yarrowia lipolytica. Yeast 9:507–517

    Google Scholar 

  • Osumi T, Tsukamoto T, Hata S, Yokota S, Miura S, Fujiki Y, Hijikata M, Miyazawa S, Hashimoto T (1991) Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Commun 186:947–954

    Google Scholar 

  • Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936–4948

    Google Scholar 

  • Roscher AA, Hoefler S, Hoefler G, Paschke E, Paltauf F, Moser A, Moser H (1989). Genetic and phenotypic heterogeneity in disorders of peroxisome biogenesis: a complementation study involving cell lines from 19 patients. Pediatr Res 26:67–72

    Google Scholar 

  • Santos MJ, Imanaka T, Shio H, Lazarow PB (1988a) Peroxisomal integral membrane proteins in control and Zellweger fibroblasts. J Biol Chem 263:10502–10509

    Google Scholar 

  • Santos MJ, Imanaka T, Shio H, Small GM, Lazarow PB (1988b) Peroxisomal membrane ghosts in Zellweger syndrome: aberrant organelle assembly. Science 239:1536–1538

    Google Scholar 

  • Simon M, Binder M, Adam G, Hartig A, Ruis H (1992) Control of peroxisome proliferation in Saccharomyces cerevisiae by ADR1, SNF1 (CAT1, CCR1) and SNF4 (CAT3). Yeast 8:303–309

    Google Scholar 

  • Subramani S (1993) Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol 9:445–478

    Google Scholar 

  • Sulter GJ, Vrieling EG, Harder W, Veenhuis M (1993a) Synthesis and subcellular location of peroxisomal membrane protein in a peroxisome-deficient mutant of the yeast Hansenula polymorpha. EMBO J 12:2205–2210

    Google Scholar 

  • Sulter GJ, Waterham HR, Vrieling EG, Goodman JM, Veenbuis M (1993b) Expression and targeting of a 47-kDa integral peroxisomal membrane protein of Candida boidinii in wild-type and peroxisome-deficient mutant cells of Hansenula polymorpha. FEBS Lett 315:211–216

    Google Scholar 

  • Swinkels BW, Gould SJ, Bodnar AG, Rachubinski RA, Subramani S (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J 10:3255–3262

    Google Scholar 

  • Titorenko VI, Waterham HR, Haima P, Harder W, Veenhuis M (1992) Peroxisome biogenesis in Hansenula polymorpha: different mutations in genes, essential for peroxisome biogenesis, cause different peroxisomal mutant phenotypes. FEMS Microbiol Lett 95:143–148

    Google Scholar 

  • Titorenko VI, Waterham HR, Cregg JM, Harder W, Veenhuis M (1993) Peroxisome biogenesis in the yeast Hansenula polymorpha is controlled by a complex set of interacting gene products. Proc Natl Acad Sci USA 90:7470–7474

    Google Scholar 

  • Tolbert NE (1974) Isolation of subcellular organelles of metabolism on isopycnic sucrose gradients. Methods Enzymol 31:734–746

    Google Scholar 

  • Tsukamoto T, Yokota S, Fujiki Y (1990) Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes. J Cell Biol 110:651–660

    Google Scholar 

  • Ueda M, Mozaffar S, Tanaka A (1990) Catalase from Candida boidinii 2201. Methods Enzymol 188:463–465

    Google Scholar 

  • Vater CA, Raymond CK, Ekena K, Howald-Stevenson I, Stevens TH (1992) The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol 119:773–786

    Google Scholar 

  • Veale RA, Guiseppin MLF, van Eijk HMJ, Sudbery PE, Verrips CT (1992) Development of a strain of Hansenula polymorpha for the efficient expression of Guar α-galactosidase. Yeast 8:361–372

    Google Scholar 

  • Veenhuis M (1992) Peroxisome biogenesis and function in Hansenula polymorpha. Cell Biochem Function 10:175–184

    Google Scholar 

  • Veenhuis M, Harder W (1987) Metabolic significance and biogenesis of microbodies in yeasts. In: Fahimi HD, Sies H (eds) Peroxisomes in biology and medicine. Springer-Verlag, Berlin, pp 436–458

    Google Scholar 

  • Veenhuis M, Harder W (1991) Microbodies. In: Rose AH, Harrison SJ (eds) The Yeasts (vol. 4, 2nd edn.) Academic Press, London, pp 601–653

    Google Scholar 

  • Veenhuis M, van Dijken JP, Pilon SAF, Harder W (1978) Development of crystalline peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions. Arch Microbiol 117:153–163

    Google Scholar 

  • Walton PA, Gould SJ, Feramisco JR, Subramani S (1992) Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal. Mol Cell Biol 12:531–541

    Google Scholar 

  • Waterham HR, Titorenko VI, van der Klei IJ, Harder W, Veenhuis M (1992) Isolation and characterization of peroxisomal protein import (Pim-) mutants of Hansenula polymorpha. Yeast 8:961–972

    Google Scholar 

  • Waterham HR, Swaving GJ, Titorenko V, Harder W, Veenhuis M (1993) Microbody biogenesis in a temperature-sensitive peroxisome-deficient mutant of the yeast Hansenula polymorpha. EMBO J 12:4785–4794

    Google Scholar 

  • Waterham HR, Titorenko VI, Haima P, Cregg JM, Harder W, Veenhuis M (1994) The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy-and amino-terminal targeting signals. J Cell Biol 127:737–749

    Google Scholar 

  • Wendland M, Subramani S (1993) Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J Cell Biol 120:675–685

    Google Scholar 

  • Zhang JW, Luckey C, Lazarow PB (1993) Three peroxisome protein-packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis. Mol Biol Cell 4:1351–1359

    Google Scholar 

  • Zoeller RA, Raetz CRH (1986) Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly. Proc Natl Acad Sci USA 83:5170–5174

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, 97291-1000, Portland, OR, USA

    Xuqiu Tan & James M. Cregg

  2. Laboratory for Electron Microscopy, Biological Center, State University of Groningen, 9751 NN, Haren, The Netherlands

    Vladimir I. Titorenko, Ida J. van der Klei, Grietje J. Sulter, Peter Haima, Hans R. Waterham, Melchior Evers, Wim Harder & Marten Veenhuis

Authors
  1. Xuqiu Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vladimir I. Titorenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ida J. van der Klei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Grietje J. Sulter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Haima
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans R. Waterham
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Melchior Evers
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wim Harder
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Marten Veenhuis
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. James M. Cregg
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by C.P. Hollenberg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tan, X., Titorenko, V.I., van der Klei, I.J. et al. Characterization of peroxisome-deficient mutants of Hansenula polymorpha . Curr Genet 28, 248–257 (1995). https://doi.org/10.1007/BF00309784

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00309784

Key words

Search

Navigation