Protein Import into Peroxisomes: The Principles and Methods of Studying

Abstract

Peroxisomes are essential intracellular organelles that involve many metabolic processes, such as β‐oxidation of very long‐chain fatty acids and synthesis of plasmalogen and bile acids as well as generation and degradation of hydrogen peroxide. These peroxisomal functions are fulfilled by strictly and spatiotemporally regulated compartmentation of the proteins catalysing these reactions. Defects in peroxisomal protein import results in inherited peroxisome biogenesis disorders in humans. Peroxisomal matrix and membrane proteins are synthesised on free ribosomes but transported into peroxisomes by distinct pathways determined by specific targeting signals and their receptors. The mechanism by which this is achieved has been clarified by identification of many PEX genes and the products named peroxins, the essential factors for peroxisome biogenesis. This article introduces several basic methods to investigate protein import into peroxisomes.

Key Concepts

  • Peroxisome plays an essential role in various metabolic pathways.
  • Deficiency of peroxisomes in human causes severe foetal genetic diseases, peroxisome‐deficient disorders.
  • Functions and the integrity of peroxisomes are archived by proper import of both matrix and membrane proteins into peroxisomes.
  • Peroxisomal proteins are encoded by nuclear DNA, synthesised in cytosolic free ribosomes and post‐translationally imported into pre‐existing peroxisomes.
  • Peroxisomal matrix and membrane proteins are translocated into peroxisomes via different pathways in a manner dependent on their distinct targeting signals.
  • Many of peroxins encoded by PEX genes are responsible factors for peroxisome biogenesis and are involved in the import of peroxisomal proteins.
  • Various experimental approaches have elucidated the molecular mechanisms underlying peroxisome biogenesis.
  • Post‐translational import of peroxisomal proteins into isolated peroxisomes can be reproduced in vitro.
  • Semi‐permeabilised cells, in which only the plasma membrane is selectively permeabilised, are used to investigate peroxisomal protein import, as with living cells.

Keywords: peroxisomes; protein translocation; peroxisome targeting signals; import receptors; PEX genes; peroxins; peroxisome biogenesis disorders

Figure 1. Peroxisomal protein import using purified peroxisomes. Cells are homogenised to break the plasma membrane but not the organelle membranes. The homogenate, before or after removal of the cell debris and unbroken cells by low‐speed centrifugation, is loaded onto a density gradient (Nycodenz or sucrose) and centrifuged at high speed up to 100 000 × g to separate peroxisomes (yellow) from other cell components. The fractions containing peroxisomes are collected and the peroxisomes are concentrated by centrifugation. The purified peroxisomes are incubated for 30 min at 25 °C with the in vitro‐translated radiolabelled peroxisomal protein of interest (shown in red). After incubation, the peroxisomes are collected by centrifugation and analysed by SDS‐PAGE and autoradiography. Import of the radiolabelled proteins is determined first by measuring the radioactivity that spins down with the peroxisomal pellet (compare lane 2 at time 0 with lane 4 at the end of the reaction), second by measuring resistance to proteases (imported proteins are protected by the peroxisomal membrane) (lanes 5 and 6) and third by decrease in molecular weight (only in the case of some PTS2 proteins that are cleaved following import). To check that the protein does not fold into a compact protease‐resistant form without being imported, the peroxisomal membrane can be broken with detergent (e.g. Triton X‐100) before adding protease (lanes 7 and 8).
Figure 2. Peroxisomal protein import using living cells. cDNAs encoding normal or mutant proteins are cloned into appropriate plasmids to allow their expression in yeast or mammalian cells. Yeast cells are usually stably transformed, whereas mammalian cells are usually transiently transfected, following a variety of possible techniques, such as electroporation or lipofection. After culturing the cells for a few days, the cells are fixed, permeabilised and immunostained using antibodies against the protein of interest and against a peroxisomal marker protein, such as a PMP Pex14p. On occasions, an autofluorescent reporter (e.g. green fluorescent protein, GFP) can be used. Secondary immunostaining using fluorochromes of different colours (e.g. fluorescein, green; Texas red, red) enables the subcellular distribution of the expressed protein to be determined accurately. Superimposition of the green image and the red image yields a yellow colour when the fluorochromes are co‐localised. In this figure, the reporter protein GFP and GFP fused with a PTS1 (GFP‐PTS1) are expressed in normal and pex5 mutant cells (PEX5 encodes the PTS1 import receptor). Upper panels: GFP shows a diffused pattern (green) in the normal cells, indicating the cytosolic localisation, while PMP Pex14p is localised to punctate structures, peroxisomes (red). Middle panels: GFP‐PTS1 (green) is imported into peroxisomes in the normal cell, as is a PMP Pex14p (red) (note: green + red = yellow in the merged view). Lower panels: no GFP‐PTS1 is imported into peroxisomes in pex5 mutant cells; it rather remains in the cytosol. Note that PMP Pex14p remains in punctate structures, peroxisome‐membrane remnants, because pex5 mutant cells are defective in import of peroxisomal matrix proteins but not in PMPs.
Figure 3. Peroxisomal protein import using semi‐permeabilised cells. Living cultured cells are semi‐permeabilised with streptolysin O or digitonin, both of which specifically permeabilise the plasma membrane but not the organelle membranes. After washing, semi‐permeabilised cells keep the organelles intact with no cytosolic components. The import substrates are separately prepared from a variety of sources such as purified recombinant proteins, in vitro translation products and proteins expressed in cultured cells. The substrate proteins are incubated with semi‐permeabilised cells under variable conditions including various factors [e.g. demand of energy (ATP) and cytosolic factors]. After terminating the import reaction, excess substrates are washed out and peroxisomal protein import is assessed by immunostaining or immunoblotting.
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References

Agrawal G and Subramani S (2013) Emerging role of the endoplasmic reticulum in peroxisome biogenesis. Frontiers in Physiology 4: 286. DOI: 10.3389/fphys.2013.00286.

Carvalho AF, Pinto MP, Grou CP, et al. (2007) Ubiquitination of mammalian Pex5p, the peroxisomal import receptor. Journal of Biological Chemistry 282: 31267–31272. DOI: 10.1074/jbc.M706325200.

Cregg JM, Van Klei IJ, Sulter GJ, Veenhuis M and Harder W (1990) Peroxisome‐deficient mutants of Hansenula polymorpha. Yeast 6: 87–97. DOI: 10.1002/yea.320060202.

Erdmann R, Veenhuis M, Mertens D and Kunau WH (1989) Isolation of peroxisome‐deficient mutants of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the USA 86: 5419–5423. DOI: 10.1073/pnas.86.14.5419.

Fujiki Y and Lazarow PB (1985) Post‐translational import of fatty acyl‐CoA oxidase and catalase into peroxisomes of rat liver in vitro. Journal of Biological Chemistry 260: 5603–5609. DOI: 10.1146/annurev.cb.01.110185.002421.

Fujiki Y, Okumoto K, Kinoshita N and Ghaedi K (2006) Lessons from peroxisome‐deficient Chinese hamster ovary (CHO) cell mutants. Biochimica et Biophysica Acta 1763: 1374–1381. DOI: 10.1016/j.bbamcr.2006.09.012.

Fujiki Y, Yagita Y and Matsuzaki T (2012) Peroxisome biogenesis disorders: molecular basis for impaired peroxisomal membrane assembly: in metabolic functions and biogenesis of peroxisomes in health and disease. Biochimica et Biophysica Acta 1822: 1337–1342. DOI: 10.1016/j.bbadis.2012.06.004.

Fujiki Y, Okumoto K, Mukai S, Honsho M and Tamura S (2014) Peroxisome biogenesis in mammalian cells. Frontiers in Physiology 5: 307. DOI: 10.3389/fphys.2014.00307.

Francisco T, Rodrigues TA, Freitas MO, et al. (2013) A cargo‐centered perspective on the PEX5 receptor‐mediated peroxisomal protein import pathway. Journal of Biological Chemistry 288: 29151–29159. DOI: 10.1074/jbc.M113.487140.

Grou CP, Carvalho AF, Pinto MP, et al. (2008) Members of the E2D (UbcH5) family mediate the ubiquitination of the conserved cysteine of Pex5p, the peroxisomal import receptor. Journal of Biological Chemistry 283: 14190–14197. DOI: 10.1074/jbc.M800402200.

Hasan S, Platta HW and Erdmann R (2013) Import of proteins into the peroxisomal matrix. Frontiers in Physiology 4: 261. DOI: 10.3389/fphys.2013.00261.

Hettema EH, Erdmann R, van der Klei I and Veenhuis M (2014) Evolving models for peroxisome biogenesis. Current Opinion in Cell Biology 29: 25–30. DOI: 10.1016/j.ceb.2014.02.002.

Hu J, Baker A, Bartel B, et al. (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24: 2279–2303. DOI: 10.1105/tpc.112.096586.

Imanaka T, Small GM and Lazarow PB (1987) Translocation of acyl‐CoA oxidase into peroxisomes requires ATP hydrolysis but not a membrane potential. Journal of Cell Biology 105: 2915–2922. DOI: 10.1083/jcb.105.6.2915.

Kim PK and Mullen RT (2013) PEX16: a multifaceted regulator of peroxisome biogenesis. Frontiers in Physiology 4: 241. DOI: 10.3389/fphys.2013.00241.

Lazarow PB and Moser HW (1995) Disorders of peroxisome biogenesis. In: Scriver CR, Beadet AL, Sly WS and Valle D, (eds). The Metabolic and Molecular Bases of Inherited Disease, pp. 2287–3324. New York: McGraw‐Hill.

Matsuzaki T and Fujiki Y (2008) The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p‐ and Pex16p‐dependent pathway. Journal of Cell Biology 183: 1275–1286. DOI: 10.1083/jcb.200806062.

Liu X, Ma C and Subramani S (2012) Recent advances in peroxisomal matrix protein import. Current Opinion in Cell Biology 24: 484–489. DOI: 10.1016/j.ceb.2012.05.003.

Miyata N, Okumoto K, Mukai S, Noguchi M and Fujiki Y (2012) AWP1/ZFAND6 functions in Pex5 export by interacting with cys‐monoubiquitinated Pex5 and Pex6 AAA ATPase. Traffic 13: 168–183. DOI: 10.1111/j.1600-0854.2011.01298.x.

Morand OH, Allen L‐AH, Zoeller RA and Raetz CRH (1990) A rapid selection for animal cell mutants with defective peroxisomes. Biochimica et Biophysica Acta 1034: 132–141. DOI: 10.1016/0304-4165(90)90066-6.

Okumoto K, Misono S, Miyata N, et al. (2011) Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling. Traffic 12: 1067–1083. DOI: 10.1111/j.1600-0854.2011.01217.x.

Platta HW, El Magraoui F, Schlee D, et al. (2007) Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling. Journal of Cell Biology 177: 197–204. DOI: 10.1083/jcb.200611012

Small GM, Imanaka T, Shio H and Lazarow PB (1987) Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes. Molecular and Cellular Biology 7: 1848–1855. DOI: 10.1128/MCB.7.5.1848.

van der Zand A and Tabak HF (2013) Peroxisomes: offshoots of the ER. Current Opinion in Cell Biology 25: 449–454. DOI: 10.1016/j.ceb.2013.05.004.

Wanders RJA and Waterham HR (2006) Biochemistry of mammalian peroxisomes revisited. Annual Review of Biochemistry 75: 295–332. DOI: 10.1146/annurev.biochem.74.082803.133329.

Wendland M and Subramani S (1993) Cytosol‐dependent peroxisomal protein import in a permeabilised cell system. Journal of Cell Biology 120 (3): 675–685. DOI: 10.1083/jcb.120.3.675.

Williams C, van den Berg M, Sprenger RR and Distel B (2007) A conserved cysteine is essential for Pex4p‐dependent ubiquitination of the peroxisomal import receptor Pex5p. Journal of Biological Chemistry 282: 22534–22543. DOI: 10.1074/jbc.M702038200.

Yagita Y, Hiromasa T and Fujiki Y (2013) Tail‐anchored PEX26 targets peroxisomes via a PEX19‐dependent and TRC40‐independent class I pathway. Journal of Cell Biology 200: 651–666. DOI: 10.1083/jcb.201211077.

Further Reading

Fagarasanu A, Mast FD, Knoblach B and Rachubinski RA (2010) Molecular mechanisms of organelle inheritance: lessons from peroxisomes in yeast. Nature Reviews Molecular Cell Biology 11: 644–654. DOI: 10.1038/nrm2960.

Fujiki Y, Okumoto K, Mukai S and Tamura S (2014) Molecular basis for peroxisome biogenesis disorders. In: Brocard C and Hartig A, (eds). Molecular Machines Involved in Peroxisome Biogenesis and Maintenance, pp. 91–110. Wien, Austria: Springer‐Verlag.

Nagotu S, Kalel VC, Erdmann R and Platta HW (2012) Molecular basis of peroxisomal biogenesis disorders caused by defects in peroxisomal matrix protein import. Biochimica et Biophysica Acta 1822: 1326–1336. DOI: 10.1016/j.bbadis.2012.05.010.

Nordgren M and Fransen M (2014) Peroxisomal metabolism and oxidative stress. Biochimie 98: 56–62. DOI: 10.1016/j.biochi.2013.07.026.

Tabak HF, Braakman I and van der Zand A (2013) Peroxisome formation and maintenance are dependent on the endoplasmic reticulum. Annual Review of Biochemistry 82: 723–744. DOI: 10.1146/annurev-biochem-081111-125123.

Wanders RJA (2014) Metabolic functions of peroxisomes in health and disease. Biochimie 98: 36–44. DOI: 10.1016/j.biochi.2013.08.022.

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Fujiki, Yukio, Okumoto, Kanji, and Honsho, Masanori(Apr 2015) Protein Import into Peroxisomes: The Principles and Methods of Studying. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002618.pub2]