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

Abstract

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 (TA^CD) 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 (Sgt2^N) recognizes basic, α-helix-prone segments within TAs^CD. NMR and mutational analyses reveal that binding is primarily driven by electrostatic complementarity at a conserved dimeric interface on Sgt2^N. A structural model of the Sgt2^N–TA^CD complex supports this binding mode, and competition experiments demonstrate that Get5^UBL can effectively displace TAs^CD from Sgt2^N via the same surface. These findings define a dual-recognition mechanism in which both the TA^CD and TMD contribute to Sgt2 engagement, thereby reinforcing client safeguarding and suggesting a broader role for Sgt2 in TA protein targeting.