title: Each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal.
abstract: We previously reported a method, termed enzyme-mediated activation of radical sources (EMARS) for analysis of co-clustered molecules with horseradish peroxidase (HRP) fusion proteins expressed in living cells. This method is featured by radical formation of labeling reagents by HRP. In the current study, we have employed another labeling reagent, fluorescein-conjugated tyramide (FT) instead of the original arylazide compounds. Although hydrogen peroxide is required for the activation of FT, the labeling efficiency by HRP and the nonspecific reactions by endogenous enzyme(s) have been dramatically improved compared with the original fluorescein arylazide. This revised EMARS method has enabled visualization of co-clustered molecules in the endoplasmic reticulum and Golgi membranes with confocal microscopy. By using this method, we have found that GPI-anchored proteins, decay accelerating factor (DAF) and Thy-1 are exclusively co-clustered with HRP-DAFGPI and HRP-Thy1GPI, in which GPI attachment signals of DAF and Thy-1 have been connected to HRP, respectively. Furthermore, the N-glycosylation types of DAF and Thy-1 have been found to correspond to those of HRP-DAFGPI and HRP-Thy1GPI, respectively. These results indicate that each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal, and that the EMARS method can segregate individual lipid rafts.
authors.affiliation: Center for Innovate and Translational Medicine, Kochi University Medical School, Nankoku, Kochi, 783-8505, Japan.
authors.forename: Arisa
authors.initials: A
authors.lastname: Miyagawa-Yamaguchi
authors.affiliation: Department of Biochemistry, Saitama Medical University, Iruma-gun, Saitama, 350-0495, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kotani
authors.affiliation: Center for Innovate and Translational Medicine, Kochi University Medical School, Nankoku, Kochi, 783-8505, Japan. khonke@kochi-u.ac.jp.|Department of Biochemistry, Kochi University Medical School, Nankoku, Kochi, 783-8505, Japan. khonke@kochi-u.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Honke
mesh_terms: D000818:Animals; D018958:CD55 Antigens; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018958:CD55 Antigens; D008565:Membrane Proteins; D006735:Horseradish Peroxidase; D019793:Fluorescein
keywords: EMARS; Fluorescein tyramide; HRP; N-glycosylation; Visualization of co-clustering molecules