Mechanistic Bases for Privileged Capture and Unidirectional Targeting of Tail-Anchored Proteins by the Get3 ATPase

C-terminal tail-anchored membrane proteins (TAs) are targeted post-translationally to the endoplasmic reticulum (ER) in eukaryotic cells mainly through the Guided entry of tail-anchored protein (GET) pathway. Here we use biochemical and biophysical approaches to shed further mechanistic insight into how the central chaperone, the Get3 ATPase, is able to capture TA substrates in a privileged manner and provide unidirectional targeting to the ER. Specifically, we first show in Chapter 2 that Get3 dynamically samples open and closed conformations as a "protean clamp". Binding of TA substrates induces Get3 to sample more open conformations that causes Get3 to dissociate from the cytosolic regulatory Get4/5 complex, hydrolyze ATP, and become primed to interact with the Get1/2 membrane receptors. Therefore, a TA substrate acts as the switch for unidirectional targeting, transitioning Get3 from a "TA-loading mode" to a "membrane targeting mode". Next, in Chapter 3, we show that a small, conserved alpha-helical lid motif, known as α8, lining the substrate binding groove is necessary for Get3 to efficiently capture TA substrates in a privileged manner over competing off-pathway chaperones.