Bipartite Recognition of Tail-Anchored Proteins by Sgt2 Involves both the Cytosolic and Transmembrane Domains

Tail-anchored (TA) membrane proteins, defined by a single C-terminal transmembrane helix, are predominantly targeted to the endoplasmic reticulum (ER) via the post-translational GET pathway. While previous studies have characterized how chaperones shield the hydrophobic TA transmembrane domain (TMD), it remains unclear whether the cytosolic domain (TACD) contributes to recognition. Here, we show that Sgt2, the entry chaperone of the GET pathway, engages TA proteins through a bipartite mechanism: its C-terminal domain captures the TMD, while its N-terminal domain (Sgt2N) recognizes basic, α-helix-prone segments within TAsCD. NMR and mutational analyses reveal that binding is primarily driven by electrostatic complementarity at a conserved dimeric interface on Sgt2N. A structural model of the Sgt2N-TACD complex supports this binding mode, and competition experiments demonstrate that Get5UBL can effectively displace TAsCD from Sgt2N via the same surface. These findings define a dual-recognition mechanism in which both the TACD and TMD contribute to Sgt2 engagement, thereby reinforcing client safeguarding and suggesting a broader role for Sgt2 in TA protein targeting.