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, S-35- 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 S-35- Pex16p with two transmembrane segments and C- tail anchor- type S-35- Pex26p, upon incubation with S-35- Pex19p in the reaction mixtures containing isolated peroxisomes. The transported S-35- Pex16p and S-35- Pex26p were integrated into membranes as assessed by the sodium carbonate extraction method. Peroxisome- associated and partly Na2CO3- resistant S-35- Pex19p was released to the cytosolic fraction upon incubation in the absence of ATP, whereas S-35- Pex16p and S-35- Pex26p remained in the membranes. Furthermore, not only 35S- Pex19p but also 35S- Pex19p complexes each with S-35- Pex16p and S-35- Pex26p were bound to S-35- 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.