Cell 149, 397–409 (2012)

Organelles generally either proliferate from preexisting organelles and then import necessary proteins from the cytosol or are derived in full from the endoplasmic reticulum (ER). Peroxisome biogenesis seems to combine elements of both strategies, with ER-derived membranes and transport proteins responsible for importing cytosolic proteins, but the way in which these pathways work together remains controversial. Now, Van der Zand et al. demonstrate that the combined path is made possible by the separate trafficking of peroxisomal components. The authors used bimolecular fluorescence complementation in combination with mutants at various steps in the biogenesis pathway to observe assembly of proteins from the two halves of the peroxisomal translocon, the docking complex and the RING finger complex. In mutants where ER export or vesicle fusion was blocked, the docking complex proteins assembled together, as did the RING finger complex proteins, but inter-complex assembly was not observed. Biochemical isolation, coimmunoprecipitation and fluorescence experiments on the unfused, immature vesicles indicated that these protein complexes were segregated in separate vesicles; pulse-chase experiments in wild-type cells confirmed this result. The authors further showed that the separate vesicles are on-pathway intermediates; after fusion of two preperoxisomal fractions, translocon assembly allowed import of a fluorescent protein tagged with a peroxisomal targeting signal. These surprising results explain the dual biogenesis strategy and raise new questions regarding protein trafficking in the ER.