RRC ID 37059
Author Miyagawa-Yamaguchi A, Kotani N, Honke K.
Title Each GPI-anchored protein species forms a specific lipid raft depending on its GPI attachment signal.
Journal Glycoconj. J.
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.
Volume 32(7)
Pages 531-40
Published 2015-10
DOI 10.1007/s10719-015-9595-5
PII 10.1007/s10719-015-9595-5
PMID 25948169
MeSH Animals CD55 Antigens Cell Line Cell Membrane / chemistry Cell Membrane / metabolism* Endoplasmic Reticulum / metabolism Fluorescein / chemistry Glycosylation Golgi Apparatus / metabolism Horseradish Peroxidase / genetics* Horseradish Peroxidase / metabolism Membrane Microdomains / chemistry Membrane Microdomains / metabolism* Membrane Proteins / chemistry Membrane Proteins / metabolism*
IF 2.548
Times Cited 2
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells