Segregation of Lipid Rafts Revealed by the EMARS Method Using GPI-Anchored HRP Fusion Proteins

Abstract

Lipid rafts serve as a platform for important biological events such as signal transduction, cell adhesion, and protein trafficking. To elucidate the molecular mechanisms of these events, identification of interacting molecules in individual raft domains is required. We have developed a novel method termed enzymemediated 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. Recently, we have established a new version of the EMARS system, in which the EMARS reaction is catalyzed by HRP expressed by genetic engineering. In order to express HRP in lipid rafts, HRP was constructed as a GPI-anchored form (HRP-GPI). Two kinds of HRP-GPIs, in which GPI attachment signals of human decay accelerating factor and Thy-1 were separately connected to the Cterminus of HRP, were expressed in human HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these HRP-GPIs underwent different N-glycosylation and formed distinct molecular clusters.Thus, this novel approach is able to identify molecular clusters associated with particular GPI-anchored proteins, suggesting that it can segregate individual lipid raft domains.

Tag Thy Neighbour: Nanometre-Scale Insights Into Kinetoplastid Parasites With Proximity Dependent Biotinylation

“…A key advantage of APEX2 catalysed proximity biotinylation is that it is very rapid, permitting labelling times as short as 1 min to be used which has allowed dynamic processes such as G-protein coupled receptor signalling to be followed (Lobingier et al, 2017;Paek et al, 2017). If the protein of interest is a cell surface protein, horseradish peroxidase (HRP) can be used to biotinylate proximal proteins either by expressing the protein fused to HRP (Miyagawa-Yamaguchi et al, 2014), or targeting the protein with a specific HRP-conjugated antibody (Bar et al, 2018). HRP has a higher activity than APEX2, unfortunately it is not active in the cytosol which limits its application to extracellular facing proteins (Hopkins et al, 2000).…”