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.
Shibata
Hiroyuki
H
Kinetic Crystallography Research Team, Membrane Dynamics Research Group, RIKEN, Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
Kashiwayama
Yoshinori
Y
Imanaka
Tsuneo
T
Kato
Hiroaki
H
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D015153:Blotting, Western; D002474:Cell-Free System; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D009185:Myelin Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D020675:Peroxisomes / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary
C063305:ABCD3 protein, human; D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009185:Myelin Proteins; D009841:Oligonucleotides; C486345:PEX16 protein, human; C073687:PMP22 protein, human; D010455:Peptides; C087343:PEX19 protein, human; D005982:Glutathione Transferase