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.
Highlights
Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and Src-family kinases
We demonstrate that molecular clusters associated with distinct horseradish peroxidase (HRP)-GPIs, in which the GPI attachment signals derived from human decay accelerating factor (DAF) and Thy-1 were separately connected to the C-terminus of HRP, are different from each other
Doxycycline was added to the culture medium (+Dox), HRP was robustly expressed on the cell surface in both HRP-GPI cases in an immunocytochemistry analysis (Figure 2B)
Summary
Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and Src-family kinases. Their sizes are small ranging mostly between 5 and 20 nm in resting cells, but could be larger on the order of a micron upon stimulation [1,2]. Heterogeneity of membrane microdomains is demonstrated in the previous study using freeze-fracture immunolabeling electron microscopy, in which different types of glycosphingolipids are found to reside in different domains [9] It remains to be elucidated whether distinct molecules are co-clustered with the different types of glycosphingolipids, the different raft domains contain common raft-associated molecules such as cholesterol, actin filament and Src-family kinases [10]
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