SUMMARYTail 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.
