Pleiner, T.*, Hazu, M.*, Pinton Tomaleri, G.*, Nguyen, V.N., Januszyk, K. and Voorhees, R.M. (2023) A selectivity filter in the ER membrane protein complex limits protein misinsertion at the ER. J. Cell. Biol. 222 e202212007
Cellular compartmentation requires stringent sorting of thousands of new proteins to their correct destination. Many membrane proteins don’t contain cleavable zip codes (signals) and are instead sorted based on subtle sequence differences! Tail-anchored proteins (TAs) are a good model, since they contain only a single C-terminal TMD followed by a short (< 50 residues) polar domain. The charge of the polar domain differs between mitochondrial (positive) and ER TAs (neutral or negative). Since the 90's we know that single charge swaps can re-direct TAs between both compartments, yet the mechanistic basis for this remained elusive. We now discovered that the machinery that inserts TAs into the ER membrane contains a positively charged selectivity filter that discriminates against mito TAs using charge repulsion. The ER membrane protein complex (EMC) ‘insertase’ needs to translocate the polar domain of a TA across the bilayer to insert the TMD. Using an improved cryo-EM structure, extensive mutagenesis and site-specific crosslinking we could map the path of a TA into the membrane. The EMC positions conserved positive charges at the entrance to the membrane to reject TAs with positively charged polar domains. Engineered EMCs with negative charges at this position show increased misinsertion of mito TAs into the ER membrane. This selectivity filter therefore limits Mito TA misinsertion into the ER membrane!