A protean clamp guides membrane targeting of tail-anchored proteins

Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3•TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating “protean clamp” that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3•Get4/5 disassembly, and inhibit the ER targeting of the Get3•TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.