The guided entry of tail-anchored proteins (GET) pathway ensures accurate delivery and insertion of a functionally diverse class of membrane proteins known as tail-anchored proteins (TAs), into the endoplasmic reticulum (ER) membrane. In Saccharomyces cerevisiae , newly-synthesized TAs are first captured by Sgt2 and then transferred to the targeting factor Get3, a process facilitated by the scaffold protein complex Get4/5. Previous studies suggest that Get4/5 promotes TA transfer by increasing lateral access for substrate entry and positioning Sgt2 nearby. However, how Get4/5 disengages from TA-loaded Get3 following the TA transfer, an event that is necessary before TA delivery to the Get1/2 insertase at the ER membrane, remains unclear. To investigate this post-transfer state, we determined the cryogenic-electron microscopy (cryo-EM) structure of the Get3–4/5 complex bound to a TA substrate and complemented these data with protein-protein interaction assays. Our structure shows that the TA cargo is positioned within Get3’s substrate binding chamber while Get3 remains associated with Get4/5. We observe that while TA binding does not cause Get4/5 release, it increases the conformational flexibility of Get4/5 within the complex, evidenced by multiple conformations of Get4/5 relative to Get3 and reduced contacts between the two proteins compared to the TA-free structure. These features suggest that the captured structure represents a transitional state in which Get4/5 is poised for dissociation. Complementary interaction assays show that efficient and complete dissociation of Get4/5 from TA-loaded Get3 requires engagement with the cytosolic domains of Get1/2. Together, these findings provide a structural snapshot of how the TA loading reconfigures the Get3-4/5 complex to prime TA-bound Get3 for downstream handoff to Get1/2 at the ER membrane.
Granados-Villanueva et al. (Sun,) studied this question.