Development of high-resolution pulse-chase method by use of click chemical reaction

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Cells cultured with precursor molecules bearing an alkyl or an azido group were quick-frozen and freeze-fracture replicas were subjected to click chemical reaction to label membrane lipids and proteins for electron microscopy. The labeling for phosphatidylcholine increased in a time-dependent manner after the precursor treatment. In budding yeast, the labeling density in the endoplasmic reticulum, nuclear membrane, mitochondrion, and vacuole was equivalent in the two membrane leaflets, whereas that in the plasma membrane showed clear asymmetry.