Abstract
Phosphatidylcholine (PtdCho) is a major membrane phospholipid synthesized in the endoplasmic reticulum. Here, we provide a protocol using electron microscopy to localize PtdCho that is newly synthesized by the Kennedy pathway in yeast cells. The protocol consists of the administration of a clickable alkyne-containing choline analog to cells, quick-freezing, freeze-fracture replica preparation, conjugation of biotin-azide by click chemical reaction, and immunogold labeling. This protocol can be used to determine quantitatively to which membrane leaflets newly synthesized PtdCho is incorporated. For complete details on the use and execution of this protocol, please refer to Orii et al. (2021).