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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Tail-anchored (TA) proteins are crucial for cellular proteostasis, but their hydrophobic transmembrane domains can lead to mistargeting.
• Mitochondrial TA proteins are often mislocalized to the endoplasmic reticulum (ER) and interact with the ER membrane protein complex (EMC).

Purpose of the Study:

• To elucidate the mechanism by which the EMC discriminates and sorts TA proteins.
• To map the insertion pathway of TA proteins into the ER membrane via the EMC.

Main Methods:

• Utilized an improved structural model of the human EMC.
• Employed site-specific crosslinking and mutagenesis to analyze TA protein interactions and membrane insertion.

Main Results:

• Mapped the TA protein insertion pathway through the EMC, from cytosolic capture to membrane insertion via a hydrophilic vestibule.
• Identified a selectivity filter at the vestibule entrance, composed of positively charged residues, that repels mitochondrial TA proteins.
• Demonstrated that this filter also retains positively charged soluble domains of multipass proteins, enforcing the 'positive-inside' rule.

Conclusions:

• The EMC employs a charge-based selectivity filter to ensure correct TA protein localization and membrane insertion.
• This mechanism prevents protein misinsertion, protects cellular compartment integrity, and provides a biochemical basis for charge-based protein sorting.