Peroxisomes pp 213-219 | Cite as

Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact Mammalian Cell System

  • Kanji Okumoto
  • Masanori Honsho
  • Yuqiong Liu
  • Yukio FujikiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1595)


Peroxisomes are essential intracellular organelles that catalyze a number of essential metabolic pathways including β-oxidation of very long chain fatty acids, synthesis of plasmalogen, bile acids, and generation and degradation of hydrogen peroxide. These peroxisomal functions are accomplished by strictly and spatiotemporally regulated compartmentalization of the enzymes catalyzing these reactions. Defects in peroxisomal protein import result in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes and transported to peroxisomes in a manner dependent on their specific targeting signals and their receptors. Peroxisomal protein import can be analyzed using a semi-intact assay system, in which targeting efficiency is readily monitored by immunofluorescence microscopy. Furthermore, cytosolic factors required for peroxisomal protein import can be manipulated, suggesting that the semi-intact system is a useful and convenient system to uncover the molecular mechanisms of peroxisomal protein import.

Key words

Import receptors Peroxisome Peroxisome targeting signals Protein import Semi-intact cells 



This work was supported in part by Grants-in-Aid for Scientific Research (24247038, 25112518, 25116717, 26116007, and 15K14511 to Y.F.; 26440032 to K.O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from the Takeda Science Foundation, the Naito Foundation, and the Japan Foundation for Applied Enzymology.


  1. 1.
    Fujiki Y (2000) Peroxisome biogenesis and peroxisome biogenesis disorders. FEBS Lett 476:42–46CrossRefPubMedGoogle Scholar
  2. 2.
    Gould SJ, Valle D (2000) Peroxisome biogenesis disorders: genetics and cell biology. Trends Genet 16:340–345CrossRefPubMedGoogle Scholar
  3. 3.
    Waterham HR, Ferdinandusse S, Wanders RJA (2015) Human disorders of peroxisome metabolism and biogenesis. Biochim Biophys Acta 1863:922–933CrossRefPubMedGoogle Scholar
  4. 4.
    Fujiki Y, Okumoto K, Mukai S, Honsho M, Tamura S (2014) Peroxisome biogenesis in mammalian cells. Front Physiol 5: Article 307Google Scholar
  5. 5.
    Yagita Y, Hiromasa T, Fujiki Y (2013) Tail-anchored PEX26 targets peroxisomes via a PEX19-dependent and TRC40-independent class I pathway. J Cell Biol 200:651–666CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Matsuzaki T, Fujiki Y (2008) The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway. J Cell Biol 183:1275–1286CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Liu X, Ma C, Subramani S (2012) Recent advances in peroxisomal matrix protein import. Curr Opin Cell Biol 24:1–6CrossRefGoogle Scholar
  8. 8.
    Hasan S, Platta HW, Erdmann R (2013) Import of proteins into the peroxisomal matrix. Front Physiol 4: Article 261Google Scholar
  9. 9.
    Fujiki Y, Rachubinski RA, Mortensen RM, Lazarow PB (1985) Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate. Biochem J 226:697–704CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Small GM, Imanaka T, Shio H, Lazarow PB (1987) Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes. Mol Cell Biol 7:1848–1855CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Gouveia AM, Guimaraes CP, Oliveira ME, Reguenga C, Sa-Miranda C, Azevedo JE (2003) Characterization of the peroxisomal cycling receptor, Pex5p, using a cell-free in vitro import system. J Biol Chem 278:226–232CrossRefPubMedGoogle Scholar
  12. 12.
    Platta HW, Grunau S, Rosenkranz K, Girzalsky W, Erdmann R (2005) Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol. Nat Cell Biol 7:817–822CrossRefPubMedGoogle Scholar
  13. 13.
    Miyata N, Fujiki Y (2005) Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export. Mol Cell Biol 25:10822–10832CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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 105:2915–2922CrossRefPubMedGoogle Scholar
  15. 15.
    Wendland M, Subramani S (1993) Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J Cell Biol 120:675–685CrossRefPubMedGoogle Scholar
  16. 16.
    Terlecky SR, Legakis JE, Hueni SE, Subramani S (2001) Quantitative analysis of peroxisomal protein import in vitro. Exp Cell Res 263:98–106CrossRefPubMedGoogle Scholar
  17. 17.
    Legakis JE, Terlecky SR (2001) PTS2 protein import into mammalian peroxisomes. Traffic 2:252–260CrossRefPubMedGoogle Scholar
  18. 18.
    Liu Y, Yagita Y, Fujiki Y (2016) Assembly of peroxisomal membrane proteins via the direct Pex19p-Pex3p pathway. Traffic 17:433–455CrossRefPubMedGoogle Scholar
  19. 19.
    Nashiro C, Kashiwagi A, Matsuzaki T, Tamura S, Fujiki Y (2011) Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, to Pex26p on peroxisome membrane. Traffic 12:774–788CrossRefPubMedGoogle Scholar
  20. 20.
    Fujiki Y, Hubbard AL, Fowler S, Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102CrossRefPubMedGoogle Scholar
  21. 21.
    Mukai S, Fujiki Y (2006) Molecular mechanisms of import of peroxisome-targeting signal type 2 (PTS2) proteins by PTS2 receptor Pex7p and PTS1 receptor Pex5pL. J Biol Chem 281:37311–37320CrossRefPubMedGoogle Scholar
  22. 22.
    Matsuzono Y, Matsuzaki T, Fujiki Y (2006) Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation. J Cell Sci 119:3539–3550CrossRefPubMedGoogle Scholar
  23. 23.
    Shimizu N, Itoh R, Hirono Y, Otera H, Ghaedi K, Tateishi K, Tamura S, Okumoto K, Harano T, Mukai S, Fujiki Y (1999) The peroxin Pex14p: cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis. J Biol Chem 274:12593–12604CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Kanji Okumoto
    • 1
  • Masanori Honsho
    • 2
  • Yuqiong Liu
    • 3
  • Yukio Fujiki
    • 2
    Email author
  1. 1.Department of Biology, Faculty of SciencesKyushu UniversityFukuokaJapan
  2. 2.Medical Institute of Bioregulation, Kyushu UniversityFukuokaJapan
  3. 3.Graduate School of Systems Life SciencesKyushu University Graduate SchoolFukuokaJapan

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