RRC ID 68823
著者 Miyagawa-Yamaguchi A, Kotani N, Honke K.
タイトル Expressed glycosylphosphatidylinositol-anchored horseradish peroxidase identifies co-clustering molecules in individual lipid raft domains.
ジャーナル PLoS One
Abstract Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains.
巻・号 9(3)
ページ e93054
公開日 2014-1-1
DOI 10.1371/journal.pone.0093054
PII PONE-D-13-28761
PMID 24671047
PMC PMC3966864
MeSH CD55 Antigens / metabolism Free Radicals / metabolism GPI-Linked Proteins / genetics GPI-Linked Proteins / metabolism* Glycosylation HeLa Cells Horseradish Peroxidase / genetics Horseradish Peroxidase / metabolism* Humans Membrane Microdomains / metabolism* Protein Transport Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Thy-1 Antigens / metabolism
IF 2.74
リソース情報
ヒト・動物細胞 293T(RCB2202)