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Published August 2023 | Published
Journal Article Open

A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER


Tail-anchored (TA) proteins play essential roles in mammalian cells, and their accurate localization is critical for proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TA proteins to the ER, where they are delivered to the insertase, the ER membrane protein complex (EMC). Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA protein from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule. Positively charged residues at the entrance to the vestibule function as a selectivity filter that uses charge-repulsion to reject mitochondrial TA proteins. Similarly, this selectivity filter retains the positively charged soluble domains of multipass substrates in the cytosol, thereby ensuring they adopt the correct topology and enforcing the "positive-inside" rule. Substrate discrimination by the EMC provides a biochemical explanation for one role of charge in TA protein sorting and protects compartment integrity by limiting protein misinsertion.

Additional Information

© 2023 Pleiner et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). We thank Songye Chen and Oliver Clarke for technical assistance, all members of the Voorhees lab for thoughtful discussion, and Alina Guna for critical reading of the manuscript. We thank Pamela Bjorkman for access to her lab's cell sorter, as well as the Caltech Flow Cytometry facility and the Caltech Cryo-EM facility. Cryo-electron microscopy was performed in the Beckman Institute Center for TEM at Caltech, and data was processed using the Caltech High Performance Cluster, supported by a grant from the Gordon and Betty Moore Foundation. This work was supported by: the Heritage Medical Research Institute (R.M. Voorhees), the National Institutes of Health's National Institute of General Medical Sciences DP2GM137412 (R.M. Voorhees), the Deutsche Forschungsgemeinschaft (T. Pleiner), and the Tianqiao and Chrissy Chen Institute (T. Pleiner, M. Hazu). Data availability: The data reported in this work are available in the published article and its online supplemental material. The atomic coordinates and cryo-EM maps have been deposited and openly available in the Protein Data Bank under accession code PDB 8S9S and in the Electron Microscopy Data Bank under accession codes EMDB-40245 (nine-subunit map), EMDB-40246 (consensus map), and EMDB-40247 (eight-subunit map). Author contributions: T. Pleiner, M. Hazu, G. Pinton Tomaleri, and R.M. Voorhees conceived the study. T. Pleiner, M. Hazu, and G. Pinton Tomaleri performed most of the experiments and analysis with assistance from V.N. Nguyen and K. Januszyk. T. Pleiner, M. Hazu, and R.M. Voorhees wrote the manuscript with input from all authors. T. Pleiner, M. Hazu, and G. Pinton Tomaleri contributed equally to this paper. Disclosures: R.M. Voorhees reported personal fees from Gate Biosciences and grants from Gate Biosciences outside the submitted work. R.M. Voorhees and G. Pinton Tomaleri are consultants for Gates Biosciences, and R.M. Voorhees is an equity holder. No other disclosures were reported.

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August 22, 2023
December 22, 2023