Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway

Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3-effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.