A selectivity filter in the EMC limits protein mislocalization to the ER
Abstract
Tail anchored proteins (TAs) play essential roles at both the ER and mitochondria, and their accurate localization is critical to proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TAs to the ER, where they are delivered to the ER membrane protein complex (EMC). We showed that the EMC directly contributes to sorting fidelity of mitochondrial TAs and multipass substrates that contain positively charged soluble domains. Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA 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 TAs. Substrate discrimination by the EMC provides a biochemical explanation for one role of charge in TA sorting and protects compartment identity by limiting protein misinsertion.
Additional Information
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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 (RMV), the NIH's National Institute Of General Medical Sciences DP2GM137412 (RMV), the Deutsche Forschungsgemeinschaft (TP), and the Tianqiao and Chrissy Chen Institute (TP, MH). Competing Interest Statement. RMV and GPT are consultants for Gates Biosciences, and RMV is an equity holder.Attached Files
Submitted - 2022.11.29.518402v1.full.pdf
Files
Name | Size | Download all |
---|---|---|
md5:626fc8500b55d11ba2d6903c7376e98c
|
10.4 MB | Preview Download |
Additional details
- Eprint ID
- 120172
- Resolver ID
- CaltechAUTHORS:20230316-182800000.62
- Gordon and Betty Moore Foundation
- Heritage Medical Research Institute
- NIH
- DP2GM137412
- Deutsche Forschungsgemeinschaft (DFG)
- Tianqiao and Chrissy Chen Institute for Neuroscience
- Created
-
2023-03-17Created from EPrint's datestamp field
- Updated
-
2023-03-17Created from EPrint's last_modified field
- Caltech groups
- Heritage Medical Research Institute, Tianqiao and Chrissy Chen Institute for Neuroscience, Division of Biology and Biological Engineering