AbstractThe correct targeting and insertion of tail-anchored (TA) integral membrane proteins (IMP) is critical for cellular homeostasis. The mammalian protein SGTA, and its fungal homolog Sgt2 (Sgt2/A), binds hydrophobic clients and is the entry point for targeting of ER-bound TA IMPs. Here we reveal molecular details that underlie the mechanism of Sgt2/A binding to TA clients. We establish that the Sgt2/A C-terminal region is conserved but flexible, sufficient for client binding, and has

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... onal and structural similarity to the DP domains of Sti1. A molecular model for Sgt2/A-C reveals a helical hand forming a hydrophobic groove, consistent with a higher affinity for TA clients with hydrophobic faces and a minimal length of 11 residues. Finally, we show that a hydrophobic face metric improves the predictions for TA localization in vivo. The structure and binding mechanism positions Sgt2/A into a broader class of helical-hand domains that reversibly bind hydrophobic clients.