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Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

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, they entirely depend on the import of target proteins to carry out their functions within peroxisomes. While the process of cargo/receptor recognition is well understood, knowledge about the molecular mechanisms of the subsequent translocation steps, including cargo release and receptor recycling, is lacking behind. Here, we provide structural and functional evidence on the role of Pex8 in these processes. First, we show that Pex8 in yeast is essential for peroxisomal cargo translocation, irrespective of the mechanism of receptor/cargo recognition. Next, we reveal that Pex8 binds through an irregular twelvefold HEAT repeat array to a short three-helical bundle within the otherwise unfolded N-terminal domain of the Pex5 receptor. Impairing this interaction abolishes peroxisomal protein translocation. It is complemented by a secondary autonomous Pex8 cargo-like interaction site with the C-terminal domain of Pex5, thus generating a bipartite interaction between the two proteins. 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 in-depth insight into the transition from cargo release into peroxisomes to receptor recycling, which is essential to uncover the overall process of the peroxisomal cargo translocation.

Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (peroxins/PEXs) cause devastating disease. PEX7 binds newly synthesized proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this import pathway remain enigmatic. Utilizing in vitro assays, yeast, and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates PTS2-protein import by binding PEX7 and stabilizing its interaction with PTS2 cargo. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess a KPWE motif necessary for PEX7 binding. Sequential binding of PEX7 to this motif in PEX39 and PEX13 provides a novel paradigm for how PTS2 cargo engage the translocation machinery. Collectively, our work uncovers an ancient and functionally important relationship among PEX39, PEX7, and PEX13, offering insights that will advance our understanding of peroxisomal biogenesis and disease.

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the membrane docking complex, composed of Pex14p, Pex17p and Pex13p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles are however unclear, mainly because structures of higher order assemblies are still lacking. Here, using an integrative approach, we provide the first structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20 nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding-sites on both membrane sides. The long rod facing the cytosol is mainly formed by the coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.