Peroxisomes are essential cellular organelles that enable the sequestered execution of a broad range of metabolic processes. Due to the lack of an internal protein synthesis machinery, peroxisomes depend on the import of target proteins to carry out their functions. For a long time, cargo-induced transient pore formation was considered the most plausible import model. However, recent findings have led to a paradigm shift suggesting the existence of a constitutive nuclear pore-like import channel 1 and a separate export channel for receptor recycling. 2 Our work closes a missing gap, by providing evidence for key events during the transition of import and export: release of cargoes into the peroxisomal lumen from the primary shuttling receptor Pex5 and the initiation of receptor recycling, involving the enigmatic importomer component Pex8. 3 First, we show that Pex8 is essential for peroxisomal cargo translocation, irrespective of the mechanism of receptor/cargo recognition. Next, we reveal by applying an integrative structural biology approach how Pex8 binds through an irregular 12-fold HEAT repeat array to a short three-helical bundle within the otherwise unfolded N-terminal domain of Pex5. Finally, we show that impairing the interaction between Pex5 and Pex8 abolishes peroxisomal protein translocation, thus demonstrating the essential role of Pex8 in this process. Our data support a model in which Pex5/Pex8 complex formation allows assembly with the peroxisomal Pex2/Pex10/Pex12 E3-ubiquitin ligase complex to initiate recycling of the receptor. In summary, our findings provide insight into the transition from cargo release into peroxisomes to receptor recycling, which is crucial for comprehending the overall process of peroxisomal cargo translocation. 1 Gao et al., (2002). Science , 378, eadf3971. 2 Feng et al., (2022). Nature , 607, 374-380. 3 Ekal et al., (2025). bioRxiv , 2025.08.30.673231.
Matthias Wilmanns (Sun,) studied this question.