In Vitro Transport of Membrane Proteins to Peroxisomes by Shuttling Receptor Pex19p*

The peroxin Pex19p comprising 299 amino acids functions in peroxisomal membrane assembly. We here developed a cell-free system for transport of membrane proteins to peroxisomes. Pex19p interacts with multiple membrane peroxins, including other membrane biogenesis peroxins, Pex16p and Pex26p, involved in matrix protein import. Cell-free synthesized, 35S-labeled Pex19p was targeted to subcellular fractions containing peroxisomes from Chinese hamster ovary-K1 cells as well as peroxisomes isolated from rat liver in an ATP-dependent manner. Such translocation was also reproduced with in vitro synthesized 35S-Pex16p with two transmembrane segments and C-tail anchor-type 35S-Pex26p, upon incubation with 35S-Pex19p in the reaction mixtures containing isolated peroxisomes. The transported 35S-Pex16p and 35S-Pex26p were integrated into membranes as assessed by the sodium carbonate extraction method. Peroxisome-associated and partly Na2CO3-resistant 35S-Pex19p was released to the cytosolic fraction upon incubation in the absence of ATP, whereas 35S-Pex16p and 35S-Pex26p remained in the membranes. Furthermore, not only 35S-Pex19p but also 35S-Pex19p complexes each with 35S-Pex16p and 35S-Pex26p were bound to 35S-Pex3p in vitro. Together, these results strongly suggested that Pex19p translocates the membrane peroxins from the cytosol to peroxisomes in an ATP- and Pex3p-dependent manner and then shuttles back to the cytosol.

[1]  I. Vastiau,et al.  Analysis of human Pex19p's domain structure by pentapeptide scanning mutagenesis. , 2005, Journal of molecular biology.

[2]  K. Rosenkranz,et al.  Structural and functional analysis of the interaction of the AAA‐peroxins Pex1p and Pex6p , 2004, The FEBS journal.

[3]  H. Kato,et al.  Domain Architecture and Activity of Human Pex19p, a Chaperone-like Protein for Intracellular Trafficking of Peroxisomal Membrane Proteins* , 2004, Journal of Biological Chemistry.

[4]  Stephan Lorenzen,et al.  Peroxisomal membrane proteins contain common Pex19p-binding sites that are an integral part of their targeting signals. , 2004, Molecular biology of the cell.

[5]  I. Vastiau,et al.  Potential Role for Pex19p in Assembly of PTS-Receptor Docking Complexes* , 2004, Journal of Biological Chemistry.

[6]  S. Gould,et al.  PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins , 2004, The Journal of cell biology.

[7]  S. Gould,et al.  PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins , 2004, The Journal of cell biology.

[8]  P. Lazarow,et al.  Peroxisome biogenesis: advances and conundrums. , 2003, Current opinion in cell biology.

[9]  S. Tamura,et al.  The pathogenic peroxin Pex26p recruits the Pex1p–Pex6p AAA ATPase complexes to peroxisomes , 2003, Nature Cell Biology.

[10]  Y. Fujiki,et al.  The Membrane Biogenesis Peroxin Pex16p , 2002, The Journal of Biological Chemistry.

[11]  K. Ghaedi,et al.  Intracellular Localization, Function, and Dysfunction of the Peroxisome-targeting Signal Type 2 Receptor, Pex7p, in Mammalian Cells* , 2002, The Journal of Biological Chemistry.

[12]  Y. Fujiki,et al.  Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import , 2002, Molecular and Cellular Biology.

[13]  J. Gärtner,et al.  Two Different Targeting Signals Direct Human Peroxisomal Membrane Protein 22 to Peroxisomes* , 2002, The Journal of Biological Chemistry.

[14]  C. Brees,et al.  Human Pex19p Binds Peroxisomal Integral Membrane Proteins at Regions Distinct from Their Sorting Sequences , 2001, Molecular and Cellular Biology.

[15]  Y. Fujiki,et al.  Hsp70 regulates the interaction between the peroxisome targeting signal type 1 (PTS1)-receptor Pex5p and PTS1. , 2001, The Biochemical journal.

[16]  S. Gould,et al.  Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins , 2001, The Journal of cell biology.

[17]  S. Subramani,et al.  The Human Peroxisomal Targeting Signal Receptor, Pex5p, Is Translocated into the Peroxisomal Matrix and Recycled to the Cytosol , 2001, Cell.

[18]  S. Gould,et al.  The genetics of peroxisome biogenesis. , 2000, Annual review of genetics.

[19]  K. Okumoto,et al.  Molecular Anatomy of the Peroxin Pex12p , 2000, The Journal of Biological Chemistry.

[20]  K. Ghaedi,et al.  The Mammalian Peroxin Pex5pL, the Longer Isoform of the Mobile Peroxisome Targeting Signal (PTS) Type 1 Transporter, Translocates the Pex7p·PTS2 Protein Complex into Peroxisomes via Its Initial Docking Site, Pex14p* , 2000, The Journal of Biological Chemistry.

[21]  Y. Fujiki,et al.  Peroxisome biogenesis and peroxisome biogenesis disorders , 2000, FEBS letters.

[22]  W. B. Snyder,et al.  The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane , 2000, The Journal of cell biology.

[23]  K. Ghaedi,et al.  The peroxin pex3p initiates membrane assembly in peroxisome biogenesis. , 2000, Molecular biology of the cell.

[24]  P. Landgraf,et al.  Human adrenoleukodystrophy protein and related peroxisomal ABC transporters interact with the peroxisomal assembly protein PEX19p. , 2000, Biochemical and biophysical research communications.

[25]  S. South,et al.  Pex19 Binds Multiple Peroxisomal Membrane Proteins, Is Predominantly Cytoplasmic, and Is Required for Peroxisome Membrane Synthesis , 2000, The Journal of cell biology.

[26]  M. van den Berg,et al.  Saccharomyces cerevisiae Pex3p and Pex19p are required for proper localization and stability of peroxisomal membrane proteins , 2000, The EMBO journal.

[27]  R. Wanders,et al.  Isolation, characterization and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants. , 1999, Human molecular genetics.

[28]  W. B. Snyder,et al.  Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris. , 1999, Molecular biology of the cell.

[29]  K. Ghaedi,et al.  The Peroxin Pex14p , 1999, The Journal of Biological Chemistry.

[30]  R. Wanders,et al.  Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  S. South,et al.  Peroxisome Synthesis in the Absence of Preexisting Peroxisomes , 1999, The Journal of cell biology.

[32]  Y. Suzuki,et al.  Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D. , 1998, American journal of human genetics.

[33]  R. Wanders,et al.  Newly Identified Chinese Hamster Ovary Cell Mutants Are Defective in Biogenesis of Peroxisomal Membrane Vesicles (Peroxisomal Ghosts), Representing a Novel Complementation Group in Mammals* , 1998, The Journal of Biological Chemistry.

[34]  T. Tsukamoto,et al.  Human PEX1 cloned by functional complementation on a CHO cell mutant is responsible for peroxisome-deficient Zellweger syndrome of complementation group I. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Erdmann,et al.  Pex19p, a Farnesylated Protein Essential for Peroxisome Biogenesis , 1998, Molecular and Cellular Biology.

[36]  M. Brown,et al.  PxF, a prenylated protein of peroxisomes. , 1994, The Journal of biological chemistry.

[37]  T. Hashimoto,et al.  Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal. , 1992, The Journal of biological chemistry.

[38]  T. Tsukamoto,et al.  Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant , 1991, Nature.

[39]  T. Tsukamoto,et al.  Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes , 1990, The Journal of cell biology.

[40]  H. Shio,et al.  Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes , 1982, The Journal of cell biology.

[41]  Y. Fujiki,et al.  Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum , 1982, The Journal of cell biology.