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.
Matsuzono
Yuji
Y
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
Fujiki
Yukio
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D000074401:Peroxins; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D013462:Sulfur Radioisotopes
D008074:Lipoproteins; D008565:Membrane Proteins; D000074401:Peroxins; C113138:Pex3 protein, human; D013462:Sulfur Radioisotopes; C087343:PEX19 protein, human; D000255:Adenosine Triphosphate