title: Asymmetrical distribution of choline phospholipids revealed by click chemistry and freeze-fracture electron microscopy.
abstract: Choline-containing phospholipids (Cho-PLs) are major components of all cellular membranes. We developed an electron microscopic technique to investigate the poorly understood problem of how Cho-PLs are distributed between membrane leaflets. Our method relies on generating freeze-fracture replicas of cells metabolically labeled with the choline analog, propargylcholine, followed by "click" reaction to conjugate biotin to propargylcholine head groups, and immunodetection of biotin with colloidal gold. Using this method in budding yeast, we found that, surprisingly, the Golgi and plasma membrane display a cytoplasmic leaflet-dominant asymmetry in Cho-PL distribution; in contrast, Cho-PLs are evenly distributed between the exoplasmic and cytoplasmic leaflets of other organelle membranes. In mammalian culture cells, the plasma membrane shows symmetrical Cho-PL distribution between leaflets, suggesting a fundamental difference between yeast and mammals. Our method should be expandable to other classes of lipids and will be useful for deciphering the mechanism responsible for generating lipid asymmetry in biological membranes.
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine , Nagoya 466-8550, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Iyoshi
authors.forename: Jinglei
authors.initials: J
authors.lastname: Cheng
authors.forename: Tsuyako
authors.initials: T
authors.lastname: Tatematsu
authors.forename: Sho
authors.initials: S
authors.lastname: Takatori
authors.forename: Masayasu
authors.initials: M
authors.lastname: Taki
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Adrian
authors.initials: A
authors.lastname: Salic
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Fujimoto
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D057930:Click Chemistry / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D005614:Freeze Fracturing / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D010743:Phospholipids / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D010743:Phospholipids; D002794:Choline