Domain Architecture and Activity of Human Pex19p, a Chaperone-like Protein for Intracellular Trafficking of Peroxisomal Membrane Proteins*

Pex19p is a peroxin involved in peroxisomal membrane biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pex19p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pex19p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex.

[1]  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.

[2]  Maurizio Pellecchia,et al.  The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393–507) in complex with the peptide NRLLLTG , 2003, Protein science : a publication of the Protein Society.

[3]  A. Roscher,et al.  The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis. , 2003, European journal of cell biology.

[4]  W. Voos.  A new connection: chaperones meet a mitochondrial receptor. , 2003, Molecules and Cells.

[5]  P. Mayerhofer,et al.  Two splice variants of human PEX19 exhibit distinct functions in peroxisomal assembly. , 2002, Biochemical and biophysical research communications.

[6]  Tomio Ogasawara,et al.  A bilayer cell‐free protein synthesis system for high‐throughput screening of gene products , 2002, FEBS letters.

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

[8]  J. Gärtner,et al.  Targeting elements in the amino-terminal part direct the human 70-kDa peroxisomal integral membrane protein (PMP70) to peroxisomes. , 2001, Biochemical and biophysical research communications.

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

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

[11]  A. Driessen,et al.  The structural basis of protein targeting and translocation in bacteria , 2001, Nature Structural Biology.

[12]  Zhaohui Xu,et al.  Crystal structure of the bacterial protein export chaperone SecB , 2000, Nature Structural Biology.

[13]  D. Valle,et al.  Peroxisome biogenesis disorders: genetics and cell biology. , 2000, Trends in genetics : TIG.

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

[15]  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.

[16]  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.

[17]  Y Endo,et al.  A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  H. Tabak,et al.  Import of proteins into peroxisomes. , 1999, Biochimica et biophysica acta.

[19]  U. Arnold,et al.  Quantitative protein precipitation from guanidine hydrochloride-containing solutions by sodium deoxycholate/trichloroacetic acid. , 1999, Analytical biochemistry.

[20]  K. Gorgas,et al.  Identification and characterization of the human peroxin PEX3. , 1999, European journal of cell biology.

[21]  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.

[22]  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.

[23]  R. Wanders,et al.  Lipid metabolism in peroxisomes in relation to human disease. , 1998, Molecular aspects of medicine.

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

[25]  H. Mewes,et al.  Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein. , 1997, Genomics.

[26]  H. Heid,et al.  Cytosolic factors mediate protein insertion into the peroxisomal membrane , 1997, FEBS letters.

[27]  H. Moser,et al.  A unified nomenclature for peroxisome biogenesis factors , 1996, The Journal of cell biology.

[28]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[29]  D. Darnall,et al.  Practical aspects of calculating protein secondary structure from circular dichroism spectra. , 1974, Biochemistry.

[30]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.