Precise timing of ATPase activation drives targeting of tail-anchored proteins.

The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at the extreme C terminus, presents major challenges to cellular protein targeting machineries. In eukaryotic cells, the highly conserved ATPase, guided entry of tail-anchored protein 3 (Get3), coordinates the delivery of TA proteins to the endoplasmic reticulum. How Get3 uses its ATPase cycle to drive this fundamental process remains unclear. Here, we establish a quantitative framework for the Get3 ATPase cycle and show that ATP specifically induces multiple conformational changes in Get3 that culminate in its ATPase activation through tetramerization. Further, upstream and downstream components actively regulate the Get3 ATPase cycle to ensure the precise timing of ATP hydrolysis in the pathway: the Get4/5 TA loading complex locks Get3 in the ATP-bound state and primes it for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction 100-fold. Our results establish a precise model for how Get3 harnesses the energy from ATP to drive the membrane localization of TA proteins and illustrate how dimerization-activated nucleotide hydrolases regulate diverse cellular processes.