A screen for GPI‐anchored protein localization uncovers a role for the posttranslational translocation and ER associated degradation machinery in regulating productive ER insertion

10–20% of all secreted proteins in eukaryotes are post‐translationaly modified at their C‐terminus with a glycosylphosphatidylinositol (GPI) anchor, thereby attaching otherwise soluble proteins to the cell membrane or cell wall. While a great deal is known about the biosynthesis of GPI anchors, much less is known regarding the regulatory machinery that governs proper insertion and quality control of GPI‐anchored proteins (GPI‐APs). To uncover, in an unbiased manner, what ER proteins take part in the correct processing of GPI‐APs in Saccharomyces cerevisiae, we examined the localization of RFP‐Gas1, a model GPI‐AP, on the background of mutants in all 400 ER resident proteins. One substantial group affecting GPI‐AP localization consisted of posttranslational translocation (PTT) machinery. Both bioinformatic and biochemical follow ups with other GPI‐APs showed that this family of proteins depends on the PTT pathway for correct ER insertion. Additional hits from the screen were from the ER Associated Degradation (ERAD) pathway. We could show that the ERAD machinery plays an unexpected role‐ clearing the cytosolic leaflet of the ER of mal‐inserted RFP‐Gas1. This work has shed light on the cellular machinery required for the initial insertion steps of GPI‐APs, while raising new questions regarding the dependency of GPI‐APs on the post‐translational insertion pathway.