Engineering yeast to induce the synthesis of GPI-APs with a permanent phosphoethanolamine on mannose 2 of the glycan moiety.

GPI-APs are a family of proteins attached to the plasma membrane by a glycoplipid that undergoes remodeling of the glycan and lipid structure during transport to the cell surface. We describe a protocol to induce the synthesis of a GPI-anchored protein whereby EtNP is added to Man2 but not removed. By temporally manipulating the expression of Gpi7p, the enzyme that adds EtNP to Man2, in ted1Δ dcr2Δ cells prior to the expression of a canonical GPI-AP (mNeon-Gas1p), EtNP is attached to Man2 of de novo synthesized mNeon-Gas1p and cannot be removed. This strategy provides a means to temporally and spatially track the transport of remodeling-defective GPI-APs in yeast cells. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).