Abstract
We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.
Full Text
The Full Text of this article is available as a PDF (1.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
- Berteaux-Lecellier V., Picard M., Thompson-Coffe C., Zickler D., Panvier-Adoutte A., Simonet J. M. A nonmammalian homolog of the PAF1 gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina. Cell. 1995 Jun 30;81(7):1043–1051. doi: 10.1016/s0092-8674(05)80009-1. [DOI] [PubMed] [Google Scholar]
- Borst P. Peroxisome biogenesis revisited. Biochim Biophys Acta. 1989 Jun 1;1008(1):1–13. doi: 10.1016/0167-4781(89)90163-2. [DOI] [PubMed] [Google Scholar]
- Brusca J. S., Radolf J. D. Isolation of integral membrane proteins by phase partitioning with Triton X-114. Methods Enzymol. 1994;228:182–193. doi: 10.1016/0076-6879(94)28019-3. [DOI] [PubMed] [Google Scholar]
- Crane D. I., Kalish J. E., Gould S. J. The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly. J Biol Chem. 1994 Aug 26;269(34):21835–21844. [PubMed] [Google Scholar]
- Cregg J. M., Barringer K. J., Hessler A. Y., Madden K. R. Pichia pastoris as a host system for transformations. Mol Cell Biol. 1985 Dec;5(12):3376–3385. doi: 10.1128/mcb.5.12.3376. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cunningham K., Lill R., Crooke E., Rice M., Moore K., Wickner W., Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. doi: 10.1002/j.1460-2075.1989.tb03457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Duve C., Baudhuin P. Peroxisomes (microbodies and related particles). Physiol Rev. 1966 Apr;46(2):323–357. doi: 10.1152/physrev.1966.46.2.323. [DOI] [PubMed] [Google Scholar]
- Didion T., Roggenkamp R. Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEBS Lett. 1992 Jun 1;303(2-3):113–116. doi: 10.1016/0014-5793(92)80500-g. [DOI] [PubMed] [Google Scholar]
- Dodt G., Braverman N., Wong C., Moser A., Moser H. W., Watkins P., Valle D., Gould S. J. Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet. 1995 Feb;9(2):115–125. doi: 10.1038/ng0295-115. [DOI] [PubMed] [Google Scholar]
- Eitzen G. A., Aitchison J. D., Szilard R. K., Veenhuis M., Nuttley W. M., Rachubinski R. A. The Yarrowia lipolytica gene PAY2 encodes a 42-kDa peroxisomal integral membrane protein essential for matrix protein import and peroxisome enlargement but not for peroxisome membrane proliferation. J Biol Chem. 1995 Jan 20;270(3):1429–1436. doi: 10.1074/jbc.270.3.1429. [DOI] [PubMed] [Google Scholar]
- 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]
- Faber K. N., Keizer-Gunnink I., Pluim D., Harder W., Ab G., Veenhuis M. The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEBS Lett. 1995 Jan 3;357(2):115–120. doi: 10.1016/0014-5793(94)01317-t. [DOI] [PubMed] [Google Scholar]
- Fransen M., Brees C., Baumgart E., Vanhooren J. C., Baes M., Mannaerts G. P., Van Veldhoven P. P. Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor. J Biol Chem. 1995 Mar 31;270(13):7731–7736. doi: 10.1074/jbc.270.13.7731. [DOI] [PubMed] [Google Scholar]
- Freemont P. S., Hanson I. M., Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. doi: 10.1016/0092-8674(91)90229-r. [DOI] [PubMed] [Google Scholar]
- Gietl C., Faber K. N., van der Klei I. J., Veenhuis M. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3151–3155. doi: 10.1073/pnas.91.8.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glover J. R., Andrews D. W., Subramani S., Rachubinski R. A. Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo. J Biol Chem. 1994 Mar 11;269(10):7558–7563. [PubMed] [Google Scholar]
- Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gould S. J., McCollum D., Spong A. P., Heyman J. A., Subramani S. Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly. Yeast. 1992 Aug;8(8):613–628. doi: 10.1002/yea.320080805. [DOI] [PubMed] [Google Scholar]
- Gärtner J., Moser H., Valle D. Mutations in the 70K peroxisomal membrane protein gene in Zellweger syndrome. Nat Genet. 1992 Apr;1(1):16–23. doi: 10.1038/ng0492-16. [DOI] [PubMed] [Google Scholar]
- Gärtner J., Obie C., Watkins P., Valle D. Restoration of peroxisome biogenesis in a peroxisome-deficient mammalian cell line by expression of either the 35 kDa or the 70 kDa peroxisomal membrane proteins. J Inherit Metab Dis. 1994;17(3):327–329. doi: 10.1007/BF00711820. [DOI] [PubMed] [Google Scholar]
- Hansen H., Didion T., Thiemann A., Veenhuis M., Roggenkamp R. Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet. 1992 Nov;235(2-3):269–278. doi: 10.1007/BF00279370. [DOI] [PubMed] [Google Scholar]
- Heyman J. A., Monosov E., Subramani S. Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis. J Cell Biol. 1994 Dec;127(5):1259–1273. doi: 10.1083/jcb.127.5.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Höhfeld J., Veenhuis M., Kunau W. H. PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis. J Cell Biol. 1991 Sep;114(6):1167–1178. doi: 10.1083/jcb.114.6.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kalish J. E., Theda C., Morrell J. C., Berg J. M., Gould S. J. Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome. Mol Cell Biol. 1995 Nov;15(11):6406–6419. doi: 10.1128/mcb.15.11.6406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamijo K., Kamijo T., Ueno I., Osumi T., Hashimoto T. Nucleotide sequence of the human 70 kDa peroxisomal membrane protein: a member of ATP-binding cassette transporters. Biochim Biophys Acta. 1992 Feb 11;1129(3):323–327. doi: 10.1016/0167-4781(92)90510-7. [DOI] [PubMed] [Google Scholar]
- Klein P., Kanehisa M., DeLisi C. The detection and classification of membrane-spanning proteins. Biochim Biophys Acta. 1985 May 28;815(3):468–476. doi: 10.1016/0005-2736(85)90375-x. [DOI] [PubMed] [Google Scholar]
- Kunau W. H., Beyer A., Franken T., Götte K., Marzioch M., Saidowsky J., Skaletz-Rorowski A., Wiebel F. F. Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: forward and reversed genetics. Biochimie. 1993;75(3-4):209–224. doi: 10.1016/0300-9084(93)90079-8. [DOI] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Kübrich M., Dietmeier K., Pfanner N. Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr Genet. 1995 Apr;27(5):393–403. doi: 10.1007/BF00311207. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lazarow P. B., Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol. 1985;1:489–530. doi: 10.1146/annurev.cb.01.110185.002421. [DOI] [PubMed] [Google Scholar]
- Liu H., Tan X., Russell K. A., Veenhuis M., Cregg J. M. PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import. J Biol Chem. 1995 May 5;270(18):10940–10951. doi: 10.1074/jbc.270.18.10940. [DOI] [PubMed] [Google Scholar]
- Liu H., Tan X., Veenhuis M., McCollum D., Cregg J. M. An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J Bacteriol. 1992 Aug;174(15):4943–4951. doi: 10.1128/jb.174.15.4943-4951.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCammon M. T., McNew J. A., Willy P. J., Goodman J. M. An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes. J Cell Biol. 1994 Mar;124(6):915–925. doi: 10.1083/jcb.124.6.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCollum D., Monosov E., Subramani S. The pas8 mutant of Pichia pastoris exhibits 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. 1993 May;121(4):761–774. doi: 10.1083/jcb.121.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
- Mosser J., Douar A. M., Sarde C. O., Kioschis P., Feil R., Moser H., Poustka A. M., Mandel J. L., Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature. 1993 Feb 25;361(6414):726–730. doi: 10.1038/361726a0. [DOI] [PubMed] [Google Scholar]
- Motley A., Hettema E., Distel B., Tabak H. Differential protein import deficiencies in human peroxisome assembly disorders. J Cell Biol. 1994 May;125(4):755–767. doi: 10.1083/jcb.125.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nguyen V. T., Morange M., Bensaude O. Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells. Anal Biochem. 1988 Jun;171(2):404–408. doi: 10.1016/0003-2697(88)90505-2. [DOI] [PubMed] [Google Scholar]
- Osumi T., Tsukamoto T., Hata S., Yokota S., Miura S., Fujiki Y., Hijikata M., Miyazawa S., Hashimoto T. Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Commun. 1991 Dec 31;181(3):947–954. doi: 10.1016/0006-291x(91)92028-i. [DOI] [PubMed] [Google Scholar]
- Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schnell D. J., Kessler F., Blobel G. Isolation of components of the chloroplast protein import machinery. Science. 1994 Nov 11;266(5187):1007–1012. doi: 10.1126/science.7973649. [DOI] [PubMed] [Google Scholar]
- Shani N., Watkins P. A., Valle D. PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6012–6016. doi: 10.1073/pnas.92.13.6012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimozawa N., Suzuki Y., Orii T., Moser A., Moser H. W., Wanders R. J. Standardization of complementation grouping of peroxisome-deficient disorders and the second Zellweger patient with peroxisomal assembly factor-1 (PAF-1) defect. Am J Hum Genet. 1993 Apr;52(4):843–844. [PMC free article] [PubMed] [Google Scholar]
- Spong A. P., Subramani S. Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. J Cell Biol. 1993 Nov;123(3):535–548. doi: 10.1083/jcb.123.3.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Swinkels B. W., Gould S. J., Subramani S. Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett. 1992 Jun 29;305(2):133–136. doi: 10.1016/0014-5793(92)80880-p. [DOI] [PubMed] [Google Scholar]
- Tan X., Waterham H. R., Veenhuis M., Cregg J. M. The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. J Cell Biol. 1995 Feb;128(3):307–319. doi: 10.1083/jcb.128.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terlecky S. R., Nuttley W. M., McCollum D., Sock E., Subramani S. The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal. EMBO J. 1995 Aug 1;14(15):3627–3634. doi: 10.1002/j.1460-2075.1995.tb00032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsukamoto T., Miura S., Fujiki Y. Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant. Nature. 1991 Mar 7;350(6313):77–81. doi: 10.1038/350077a0. [DOI] [PubMed] [Google Scholar]
- Tsukamoto T., Shimozawa N., Fujiki Y. Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis. Mol Cell Biol. 1994 Aug;14(8):5458–5465. doi: 10.1128/mcb.14.8.5458. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van der Leij I., Franse M. M., Elgersma Y., Distel B., Tabak H. F. 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 U S A. 1993 Dec 15;90(24):11782–11786. doi: 10.1073/pnas.90.24.11782. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wanders R. J., Heymans H. S., Schutgens R. B., Barth P. G., van den Bosch H., Tager J. M. Peroxisomal disorders in neurology. J Neurol Sci. 1988 Dec;88(1-3):1–39. doi: 10.1016/0022-510x(88)90203-1. [DOI] [PubMed] [Google Scholar]
- Waterham H. R., Titorenko V. I., Haima P., Cregg J. M., Harder W., Veenhuis M. 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. 1994 Nov;127(3):737–749. doi: 10.1083/jcb.127.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Waterham H. R., Titorenko V. I., Swaving G. J., Harder W., Veenhuis M. Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles. EMBO J. 1993 Dec;12(12):4785–4794. doi: 10.1002/j.1460-2075.1993.tb06167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wiemer E. A., Nuttley W. M., Bertolaet B. L., Li X., Francke U., Wheelock M. J., Anné U. K., Johnson K. R., Subramani S. Human peroxisomal targeting signal-1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J Cell Biol. 1995 Jul;130(1):51–65. doi: 10.1083/jcb.130.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Hoop M. J., Ab G. Import of proteins into peroxisomes and other microbodies. Biochem J. 1992 Sep 15;286(Pt 3):657–669. doi: 10.1042/bj2860657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van den Bosch H., Schutgens R. B., Wanders R. J., Tager J. M. Biochemistry of peroxisomes. Annu Rev Biochem. 1992;61:157–197. doi: 10.1146/annurev.bi.61.070192.001105. [DOI] [PubMed] [Google Scholar]