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.
A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER
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.
K. Januszyk’s current affiliation is Neomorph, Inc., San Diego, CA, USA.
- Award Id(s): DP2GM137412
Tino Pleiner, Masami Hazu, Giovani Pinton Tomaleri, Vy N. Nguyen, Kurt Januszyk, Rebecca M. Voorhees; A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER. J Cell Biol 7 August 2023; 222 (8): e202212007. doi: https://doi.org/10.1083/jcb.202212007
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