Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway. Background: Get4/5 is required for the efficient transfer of tail-anchored proteins to Get3. Results: The Get3·Get4/5 complex forms an intermediate mediated by electrostatic interactions. Conclusion: The rapid association of the Get3·Get4/5 intermediate complex is followed by a conformational change to the stable inhibited structure dominated by hydrophobic interactions. Significance: These results provide insight into the mechanism of tail-anchored protein targeting.