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Published December 2018 | Accepted Version
Journal Article Open

In vitro Assays for Targeting and Insertion of Tail‐Anchored Proteins Into the ER Membrane


Membrane proteins mediate numerous essential cellular functions. Due to the aggregation propensity of hydrophobic transmembrane domains in aqueous environments, the targeting and insertion of membrane proteins pose major challenges to cells. In the Guided Entry of Tail‐anchored protein (GET) pathway, an essential class of newly synthesized tail‐anchored proteins (TAs) are chaperoned and guided by multiple targeting factors to the endoplasmic reticulum (ER) membrane. Deciphering the molecular mechanism of this cellular process has benefitted from successful in vitro reconstitution of individual molecular events in the GET pathway with purified components. Here we describe recently developed protocols for in vitro reconstitution of functional complexes of TA substrates with their targeting factors, for monitoring the transfer of TAs between targeting factors, and for the insertion of TA into the microsomal membrane. These procedures are generally applicable to the interrogation of other post‐translational membrane protein targeting pathways.

Additional Information

© 2018 by John Wiley & Sons, Inc. Version of Record online: 25 September 2018. We thank members of the Shan lab for comments on reagent preparation. This work was supported by NIH grant GM107368, Gordon and Betty Moore Foundation Grant GBMF2939, and a fellowship from the Weston Havens Foundation to S.-o.S.

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Accepted Version - nihms970064.pdf


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August 22, 2023
October 18, 2023