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

Abstract

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.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins

“…In contrast, substrates that are poorly inserted by the EMC (i.e., those that have increased N-terminal positive charge but also likely including those with longer or more structured soluble domains) have a longer dwell time at the cytosolic vestibule of the EMC. This is consistent with their increased reliance on the cytosolic C-terminus of EMC7 that contains several conserved hydrophobic residues, previously shown to directly interact with substrates in the cytosol (Pleiner et al, 2023). These data are consistent with the model that the rate limiting step for insertion is translocation of the N-terminal soluble domain through the hydrophilic vestibule of the EMC.…”

“…Further, we verified using chemical crosslinking that CCDC47 is a direct physical interactor of the EMC and that its interaction is EMC7-dependent (Figure 4D, S5F). EMC7 is a peripheral subunit of the EMC, and as such, knockout of EMC7 does not destabilize core EMC subunits (Pleiner et al, 2023). This therefore suggests that the interaction between the EMC and CCDC47 is highly specific, as loss of a single peripheral subunit abolishes CCDC47 interaction with the EMC.…”

A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes

“…The mechanisms that Oxa1 uses for protein insertion are similar to the ER Membrane Complex (EMC), its distant homolog (Kizmaz & Herrmann, 2023). In vivo fluorescence assays were among the most important tools that helped to gain considerable insight into how EMC selects its substrates and ensures correct TMD orientation within the membrane (Wu et al , 2023; Pleiner et al , 2023; Fenech et al , 2023). We suggest that BiG Mito-Split can be an excellent tool to address these mechanisms for Oxa1.…”

ER membrane complex (EMC): Structure, functions, and roles in diseases

“…Those first TMDs efficiently inserted by the EMC, such as those featuring negative charges and short N‐terminal soluble structural domains, enter the bilayer via their hydrophilic vestibule. TMDs not promptly inserted by the EMC are transferred to other insertion enzymes, including TMCO1, and in some cases, Sec61 22,59 …”

“…Recent studies underscore the vital role of EMC in maintaining ER function, impacting processes such as protein and lipid synthesis, organelle communications, ER stress, viral infection, and various diseases 7,8,21‐26 . Given the newfound significance of EMC in biochemical processes and diseases, investigations into their structure, functions, and pathogenesis‐related studies have become an area of considerable interest.…”