Accurate protein localization is crucial to generate and to maintain cellular organization. Achieving accuracy is challenging, as the molecular signals that dictate a protein’s destination are often promiscuous. The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at its extreme C-terminus, presents major challenges to protein targeting machineries. This dissertation explores how TA capture and release are spatially and temporally regulated in the Guided Entry of Tail Anchored proteins (GET) pathway and how endoplasmic reticulum (ER) destined TAs are targeted with high fidelity.
A quantitative framework of the Get3 ATPase cycle reveals that ATP and GET pathway effector proteins specifically induce multiple conformational changes in Get3, which culminate in the ATPase activation that drives unidirectional targeting in the pathway. The Get4/5 TA loading complex locks Get3 in the ATP-bound state that is primed for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction.
Additional analyses define multiple physicochemical features that distinguish TA proteins destined to different organelles. The GET pathway selects for these features at distinct stages using mechanisms such as differential binding, induced fit, and kinetic proofreading after ATP hydrolysis by Get3. These results reveal new roles for the cochaperone Sgt2 in providing key selection filters, and provide a biological logic for the complex cascade of substrate relay events during post-translational membrane protein targeting.</p
