Research / Clinical
Summary
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Diseases/Research Topics
Signal Transduction
We are interested in peroxisome biogenesis and degradation, the mechanism of peroxisomal matrix and membrane protein import, and the intracellular movement of this organelle. Our interest in the mechanism of import stems from the fact that the transport of proteins across the peroxisomal membrane is distinct from that used for other organelles, and from the realization that many human patients with fatal peroxisomal disorders (Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and rhizomelic chondrodysplasia punctata, RCDP) fail to import one or more proteins into the peroxisome matrix1. We discovered two of the peroxisomal targeting signals (PTSs), called PTS1 and PTS2, involved in the import of proteins into the peroxisomal matrix2. We have also described several sequences called mPTSs, involved in the targeting of peroxisomal membrane proteins (PMPs)3-5.
Work in a number of model systems has led to the cloning of many PEX genes and the characterization of about 23 peroxins - proteins involved in peroxisomal protein import, biogenesis and in peroxisome segregation to daughter cells. Of these at least 13 are conserved between yeasts and humans and 10 are implicated in fatal human peroxisomal disorders1. We cloned and characterized the PTS1 receptor gene (PEX5) from P. pastoris and from humans6,7. We also cloned and characterized the P. pastoris PTS2 receptor gene (PEX7), whose human counterpart is mutated in RCDP patients8. About a dozen other peroxisome assembly (PEX) genes have been characterized in this laboratory1,2. The peroxins Pex5p, Pex7p, Pex13p, Pex14p, Pex17p play a role in matrix protein import, while Pex3p, Pex17p and Pex19p are involved in the import and assembly of PMPs in the peroxisomal membrane. Pex4p, a ubiquitin-conjugating enzyme held at the peroxisomal membrane by Pex22p, also plays a role in peroxisome biogenesis. Pex1p and Pex6p are vesicle-associated ATPases that are required for vesicle-fusion events during peroxisome biogenesis9. We have also characterized many interactions among the peroxins. Human counterparts of most of these proteins are involved in fatal disorders1.
Using cytosol- and energy-dependent mammalian in vitro import systems, we have shown a requirement for the PTS, ATP hydrolysis, cytosolic hsp70, hsp40 and Pex5p, as well as the peroxisomal membrane proteins, Pex2p and Pex14p, in the import of proteins into the peroxisomal matrix10. Using purified, recombinant components, we are utilizing the in vitro import system to elucidate the biochemical functions of all the cytosolic and membrane-associated proteins involved in the import of PTS-containing proteins. Unlike the import of proteins across other organellar membranes, protein unfolding is not essential for peroxisomal matrix protein import2.
Current work focuses on the early steps in the biogenesis of peroxisomal matrix and membrane proteins3, 11, 12, the steps and intermediates in peroxisome biogenesis1,2, the protein-protein interactions among peroxins1, peroxisome movement13, and the genetics of peroxisome degradation14. Both yeast and mammalian cells are used for the analysis of these phenomena.
Keller, G., Gould, S., DeLuca, M. and Subramani, S. (1987). Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl. Acad. Sci. USA 84:3264-3268.
Gould, S.J., Keller, G.-A. and Subramani, S. (1987). Identification of a peroxisomal targeting signal at the carboxy-terminus of firefly luciferase. J. Cell. Biol. 105:2923-2931.
Gould, S.J., Keller, G.-A. and Subramani, S. (1988). Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J. Cell Biol., 107:897-905.
Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. and Subramani, S. (1989). A conserved tripeptide sorts proteins to peroxisomes. J. Cell. Biol., 108 :1657-1664
Gould, S.J., Krisans, S. Keller, G., and Subramani, S. (1990). Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins. J. Cell Biol., 110:27-34
Gould, S.J., Keller, G.-A., Schneider, M., Howell, S.H., Garrard, L.J., Goodman, J.M., Distel, B., Tabak, H. and Subramani, S. (1990). Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J., 9:85-90.
Gould, S.J. and Subramani, S. (1991). Translocation of proteins into peroxisomes, in Intracellular Trafficking of Proteins (ed. C.J.Steer and J. Hanover), Cambridge University Press, Cambridge, England, Chapt. 20, pp 696-730.
Keller, G.-A., Krisans, S.K., Gould, S.J., Sommer, J., Wang, C.C., Schliebs, W., Kunau, W., Brody, S. and Subramani, S. (1991). Evolutionary conservation of a signal that targets proteins to peroxisomes, glyoxysomes and glycosomes. J. Cell. Biol. 114:893-904.
Subramani, S. (1991). Peroxisomal targeting signals-the beginning and the end. Curr. Sci., Indian Acad. Sci. 61:28-32.
Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991). A novel cleavable peroxisomal targeting signal at the amino terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J. 10:3255-3261.
Subramani, S. (1992). Targeting of proteins into the peroxisomal matrix. J. Memb. Biol. 125:99-106.
Walton, P.A., Gould, S.J., Feramisco, J.R. and Subramani, S. (1992). Transport of microinjected proteins intoperoxisomes of mammalian cells: Inability of Zellweger cell lines to import proteins with the SKL peroxisomal targeting signal. Mol. Cell Biol. 12:531-541.
Walton, P.A., Gould, S.J., Rachubinski, R.A., Subramani, S. and Feramisco, J.R. (1992). Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: Involvement of the peroxisomal targeting signal. J. Cell Biol., 118:499-508 .
Gould, S.J., McCollum, D., Spong, A.P., Heyman, J.A. and Subramani, S. (1992). Development of the yeast Pichia pastoris as a model organism for a genetic analysis of peroxisome assembly. Yeast, 8:613-628.
Distel, B., Gould, S.J., Voorn-Brouwer, T., van der Berg, M. Tabak, H., and Subramani, S. (1992). The carboxy-terminal tripeptide Serine-Lysine-Leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast. New Biologist 4:157-165.
Subramani, S. (1992). Mechanisms of protein transport into microbodies. In Membrane Biogenesis and Protein Targeting, New Comprehensive Biochemistry, (W. Neupert and R. Lill, eds), Elsevier, The Netherlands, Ch 17, pp 221-229.
Blattner, J., Swinkels, B., Dörsam, M., Prospero, T., Subramani, S. and Clayton, C. (1992). Glycosome assembly in trypanosomes: variations in the acceptable degeneracy of a C-terminal microbody targeting signal. J. Cell Biol., 119:1129-1136.
Swinkels, B.W., Gould, S.J. and Subramani, S. (1992). Targeting efficiencies of various permutations of the consensus C-terminal tripeptide peroxisomal targeting signal. FEBS Lett. 305:133-136.
Wendland, M. and Subramani, S. (1993). Cytosol dependent peroxisomal protein import in a permeabilized cell system. J. Cell Biol. 120:675-685
McCollum, D. M., Monosov, E. and Subramani, S. (1993) The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger Syndrome cells - The PAS8 protein binds the COOH-terminal tripeptide peroxisomal targeting signal and is a member of the TPR protein family. J. Cell Biol.121:761-774.
Subramani, S. (1993) Protein import into peroxisomes and biogenesis of the organelle. Annu. Rev. Cell Biol. 9:445-478
Wendland, M., Subramani, S. (1993) Presence of cytoplasmic factors functional in peroxisomal protein import implicates organelle-associated defects in several human peroxisomal disorders. J. Clin. Invest. 92:2462-2468.
Spong, A., Subramani, S. (1993). Cloning and characterization of PAS5: A gene required for peroxisome biogenesis in the methylotropic yeast Pichia pastoris. J. Cell Biol. 123:535-548
Glover, J. R., Andrews, D. W., Subramani, S., Rachubinski, R. A. (1994) Mutagenesis of the amino-targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids r equired for import into peroxisomes in vivo. J. Biol. Chem. 269:7558-7563.
Walton, P. A., Wendland, M., Subramani, S., Rachubinski, R. A., Welch, W. J. (1994) Involvement of 70-kD heat-shock proteins in peroxisomal import. J. Cell Biol. 125:1037-1046.
Heyman, J. A., Monosov, E., Subramani, S. (1994) Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis. J. Cell Biol. 127:1259-1273.
Wiemer, E. A. C., Subramani, S. (1994) Protein import deficiencies in human peroxisomal disorders. Mol. Genet. Med. 4:119-152.
Walton, P. A., Hill, P. E., Subramani, S. (1995) Import of stably-folded proteins into peroxisomes. Mol. Biol. Cell, 6:675-683.
Wiemer, E. A. C., Nuttley, W. M., Bertolaet, B. L., Li, X., Francke, U., Wheelock, M. J., Anne, U. K., Johnson, K. R., Subramani, S. (1995) The human PTS1 receptor restores peroxisomal protein import in cells from patients with fatal peroxisomal disorders. J. Cell Biol. 130:51-65.
Terlecky, S.R., Nuttley, W. M., McCollum, D., Sock, E., Subramani, S. (1995) The Pichia pastoris peroxisomal protein, PAS8p, is the receptor for the carboxy-terminal, tripeptide peroxisomal targeting signal. EMBO J. 14:3627-3634.
Rachubinski, R., Subramani, S. (1995) How proteins penetrate peroxisomes. Cell, 83: 525-528.
Wiemer, E. A. C., Terlecky, S. R., Nuttley, W. M., Subramani, S. (1995) Characterization of the yeast and human receptors for the carboxy-terminal, tripeptide peroxisomal targeting signal. Cold Spring Harbor Symp. Quant. Biol. Vol. LX, 637-648.
Terlecky, S. R., Wiemer, E. A. C., Nuttley, W. M. , Walton, P. A., Subramani, S. (1996) Signals, receptors and cytosolic factors involved in peroxisomal protein import, in Peroxisomes: Biology and Role in Toxicology and Disease. (eds. J. K. Reddy, T. Suga, G.P. Mannaerts, P.B. Lazarow and S. Subramani) Annals New York Acad. Sci. 804: 11-20.
Monosov, E., Wenzel, T. J., Lüers, G. H., Heyman, J. A., Subramani, S. (1996) Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells. J. Histochem. Cytochem. 44: 581-589.
Subramani, S. (1996) Convergence of model systems for peroxisome biogenesis. Curr. Opin. Cell Biol. 8: 513-518.
Wiemer, E.A.C., Lüers, G., Faber, K. N., Wenzel, T., Veenhuis, M., Subramani, S. (1996) Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. J. Biol. Chem. 271: 18973-18980.
Subramani, S. (1996). Protein translocation into peroxisomes. J. Biol. Chem. 271: 32483-32486
Terlecky, S. R., Nuttley, W. M., Subramani, S. (1996) The requisite cytosolic and membrane components of peroxisomal protein import. Experientia 52:1050-1054.
Distel, B., Erdmann, R., Gould, S.J., Blobel, G., Crane, D.I., Cregg, J.M., Dodt, G., Fujiki, Y., Goodman, J.M., Just, W. W., Kiel, J.A.K.W., Kunau, W.H., Lazarow, P.B., Mannaerts, G.P., Moser, H., Osumi, T., Rachubinski, R.A., Roscher, A., Subramani, S., Tabak, H.F., Valle, D., van der Klei, I., van Veldhoven, P.P., Veenhuis, M. (1996) A unified nomenclature for peroxisome biogenesis. J. Cell Biol. 135: 1-3.
Wiemer, E. A.C., Wenzel, T., Deerinck, T. J., Ellisman, M. H. , Subramani, S. (1997) Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules. J. Cell Biol. 136: 71-80.
Subramani, S. (1997) PEX genes on the rise. Nature Genetics, 15: 331-333.
Elgersma, Y., Kwast, L., van den Berg, M., Snyder, W.B., Distel, B., Subramani, S., Tabak, H.F. (1997). Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisomal assembly in S. cerevisiae, causes proliferation of the endoplasmic reticulum membrane. EMBO J., 16: 7326-7341.
Sakai, Y., Subramani, S. Green fluorescent protein (GFP) fluorescence through the rhodamine channel after excitation using the FITC filter set. Tech. Tips Online (http://www.elsevier.com/locate/tto), T01319, (1997).
Faber, K. N., Elgersma, Y., Heyman, J. A., Koller, A., Lüers, G. H., Nuttley, W. M., Terlecky, S. R., Wenzel, T. J., Subramani, S. (1998) Use of Pichia pastoris as a model eukaryotic system: Peroxisome biogenesis, in Methods in Molecular Biology: Pichia protocols (ed. D. R. Higgins), Humana Press, Totowa, NJ, USA, Ch. 10, pp 121-147.
Subramani, S. (1998) Components involed in peroxisome import, biogenesis, proliferation, turnover and movement. Physiol. Rev. 78: 171-188.
Faber, K. N., Heyman, J.A., Subramani, S. (1998) The AAA-family peroxins, PpPex1p and PpPex6p, interact with each other in an ATP-dependent manner and are associated with different subcellular membranous structures distinct from peroxisomes. Mol. Cell. Biol. 18: 936-943.
Elgersma, Y., Elgersma-Hooisma, M., Wenzel, T., McCaffery, J.M., Farquhar, M.G., Subramani, S. (1998) A mobile PTS2-receptor for peroxisomal protein import in Pichia pastoris. J. Cell Biol. 140:807-820.
Lüers, G.H., Advani, R., Wenzel, T., Subramani, S. (1998) The Pichia pastoris dihydroxyacetone kinase is a PTS1-containing, but cytosolic, protein that is essential for growth on methanol. Yeast, 14: 759-771.
Sakai, Y., Koller, A., Rangell, L.K., Keller, G.A., Subramani, S. (1998) Peroxisome degradation by microautophagy in Pichia pastoris: identification of specific steps and morphological intermediates. J. Cell Biol. 141: 625-636.
Fransen, M.F., Terlecky, S.R., Subramani, S. (1998) Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import. Proc. Natl. Acad. Sci. USA, 95: 8087-8092.
Subramani, S. (1998) Biogenesis of peroxisomes, in Lipid and Protein Traffic, Pathways and Molecular Mechanisms, (ed. J. A. F. Op den Kamp), NATO ASI Series, Vol. H 106, pp65-76, Springer-Verlag.
Fransen, M., Van Veldhoven, P.P., Subramani, S. (1999) Identification of peroxisomal proteins using pVI-phage display: Molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase. Biochem. J. 340: 561-568.
Snyder, W.B., Faber, K.N., Wenzel, T.J., Koller, A., Lüers, G.H., Rangell, L., Keller, G.A., Subramani, S. (1999) Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris. Mol. Biol. Cell, 10:1745-1761.
Koller, A., Spong, A.P., Lüers, G.H., Subramani, S. (1999) Analysis of the peroxisomal Acyl-CoA oxidase gene product from Pichia pastoris and determination of its targeting signal. Yeast, 15:1035-1044.
Koller, A., Snyder, W.B., Faber, K.N., Wenzel, T.J., Rangell, L., Keller, G.A., Subramani, S. (1999) Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane. J. Cell Biol., 146: 99-112.
Yamashita, H., Avraham, S., Jiang, S., London, R., Van Veldhoven, P.P., Subramani, S, Rogers, R.A., Avraham, H. (1999). Characterization of the human and murine PMP20 peroxisomal membrane proteins that exhibit antioxidant activity in vitro. J. Biol. Chem., 274: 29897-29904
Snyder, W.B., Koller, A., Choy, A.J., Johnson, M., Cregg, J.M., Rangell, L., Keller, G.A., Subramani, S. (1999). Pex17p is required for import of both peroxisome membrane and lumenal proteinsand interacts with Pex19p and the PTS-receptor docking complex in Pichia pastoris. Mol. Biol. Cell , 10: 4005-4019
Subramani, S., Koller, A., Snyder, W.B. (2000). Import of peroxisomal matrix and membrane proteins. Annu. Rev. Biochem. in press
Koller, A., Velasco, J., Subramani, S. (2000) The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris. Yeast, 16:651-656.
Snyder, W.B., Koller, A., Choy, A.J., Subramani, S. (2000) The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane. J. Cell Biol. 149: 1171-1177.
Sakai, Y., Subramani, S. (2000) Environmental response of yeast peroxisomes: aspects of organelle assembly and degradation. Cell Biochem. Biophys. in press.
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