Site-directed mutagenesis and protein expression

All polymerase chain reaction (PCR) amplification and site-directed mutagenesis, including point and deletion mutations were performed using Platinum™ Pfx or SuperFi™ DNA polymerase (Thermo Fisher Scientific). Subcloning of open reading frames (ORFs) and their derivatives into expression plasmids including pICE (a gift from Steve Jackson; Addgene plasmid #46960), pGEX-4T-1 (GE Healthcare Life Sciences), and NanoBiT® system vectors (Promega) was performed using appropriate restriction enzyme sites (Supplementary Table 1). Overlap extension PCR^{56 (link)} was carried out for replacement of the NIX tail-anchor (TA) region with the TA region of other tail-anchored proteins and the fused genes were cloned into vectors pBiT1.1-N (Promega) and pICE_V5^{33 (link)}. Primers are listed in Supplementary Table 3.