Precise Timing of ATPase Activation Drives Targeting of Tail-Anchored Proteins

Hundreds of proteins are anchored on cellular membranes by a transmembrane domain (TM) at their extreme C-terminus. These ‘tail-anchored' (TA) proteins perform numerous essential cellular functions, yet their unique topology poses fundamental challenges to their proper localization. In eukaryotic cells, the highly conserved ATPase Get3 coordinates the efficient capture and delivery of TA proteins to the endoplasmic reticulum (ER). How Get3 uses its ATPase cycles to drive this fundamental process remains elusive. Here we define the Get3 ATPase cycle and show that ATP specifically induces conformational changes in Get3 that culminate in its ATPase activation through tetramerization. This activation is inhibited by the TA protein loading complex Get4/5 and is activated by the TA substrate, ensuring the precise timing of ATP hydrolysis during targeting. Our results provide an explicit model for how Get3 harnesses the energy from ATP to spatially and temporally coordinate the membrane localization of TA proteins