Abstract
Membrane proteins interact with membrane lipids for their structural stability and proper function. However, lipid–protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology. Here, we report that amphiphilic lipid probes can be used to link membrane lipids and membrane proteins in vivo. Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea. In the erythrocyte, the cholesterol probe fluorescently tagged the anion transporter band 3 via thiourea. Tagging by the cholesterol probe, but not by the phospholipid probe, was competitive with an anion transporter inhibitor, implying the presence of a specific binding pocket for cholesterol in this ~100 kDa protein. This method could prove an effective strategy for analyzing lipid–protein interactions in vivo in the live cell membrane.
Highlights
How membrane lipids and membrane proteins interact with each other is a fundamental question in biology, as lipid–protein (LP) interactions are expected to play important roles in the cell membrane[1,2,3]
There have been reports of cholesteryl ethers with polyethylene glycol (PEG) forming liposomes[16] or micelles[17,18] in aqueous solutions. Such amphiphilic molecules can be used as cholesterol probes with no membrane permeability, e.g. fluorescein-labeled PEG-cholesterol molecules with a PEG repeat of 50 or 200 were shown to distribute to cholesterol-rich domains in the outer leaflet of the plasma membrane[18,19]
Judging from the molecular weight and the SDS-PAGE pattern, the tagged protein was identified as the anion transporter band 3, which was subsequently confirmed by LC-MS/MS analysis (Figure S1)
Summary
How membrane lipids and membrane proteins interact with each other is a fundamental question in biology, as lipid–protein (LP) interactions are expected to play important roles in the cell membrane[1,2,3]. Biochemical analyses have unveiled specific functions of membrane lipids; for example, conferring thermal stability to proteins and modulating the affinity between proteins and ligands. The quantity, activity, and subcellular localization of the corresponding protein can be detected using these approaches Such irreversible– modification approaches may be applicable to membrane lipids for detecting LP interactions. We demonstrate that the chemical tagging approach using thiourea as a reactive function can be used to link lipids and the anion transporter band 3 in the erythrocyte membrane. This approach enabled us to identify a putative binding pocket of cholesterol in a large ~100 kDa membrane protein
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