J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting client trapping and regulating the ATPase cycle. Here, we report that JDPs can further enhance targeting competence of Hsp70-bound during tail-anchored protein (TA) biogenesis. In guided-entry-of-tail–anchored pathway yeast, nascent TAs are captured cytosolic sequentially relayed to downstream chaperones, Sgt2 Get3, fo...