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Edited by James H. Hurley, University of California, Berkeley, CA, and approved October 19, 2020 (received for review May 16, 2020)
Significance
Peroxisomal matrix enzymes are synthesized in the cytosol and carry targeting signals. These are recognized and bound by an import receptor in the cytosol. The cargo-loaded receptor further binds to the “docking” complex at the peroxisomal membrane. The docking events are expected to trigger the formation of a transient pore allowing import of the cargo into the peroxisomal matrix. Here using cryoelectron microscopy complemented by native MS, cross-linking MS, SEC MALS, and immunogold labeling, we provide the structural characterization of the major components of the yeast peroxisomal docking complex. Pex14p/Pex17p assemble in a parallel 3:1 bundle arrangement to form intriguingly long flexible rods that emanate into the cytosol to recruit the cargo-loaded receptor for further translocation events.
Abstract
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.
Footnotes
↵1P.L., T.H., and D.W. contributed equally to this work.
↵2Present address: Interdisciplinary Research Center HALOmem, Institute for Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany.
↵3Present address: Protagen Protein Services GmbH, 74076 Heilbronn, Germany.
- ↵4To whom correspondence may be addressed. Email: bettina.warscheid{at}biologie.uni-freiburg.de, Ralf.Erdmann{at}rub.de, or christos.gatsogiannis{at}uni-muenster.de.
Author contributions: B.W., R.E., and C.G. designed research; P.L., T.H., D.W., B.U.K., T.J., D.V., J.M., J.B., A.S., F.D., W.G., and C.G. performed research; P.L., T.H., D.W., B.U.K., T.J., J.B., F.D., W.G., and C.G. analyzed data; and B.W., R.E., and C.G. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2009502117/-/DCSupplemental.
Data Availability.
The EM density map has been deposited in the Electron Microscopy Data Bank under accession code EMD-12047. MS raw data and result files have been deposited in the ProteomeXchange Consortium via the PRIDE repository (56) and are publicly accessible from its website with the dataset identifier PXD016304. All study data are included in the article and supporting information.
Published under the PNAS license.
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Towards the molecular architecture of the peroxisomal receptor docking complex
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