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There is much evidence to indicate that cholesterol forms lateral membranemicrodomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of h-toxin (perfringolysin O), a cholesterol-binding cytolysin, whose chemicallymodified formhas been recently shown to bind selectively to rafts. . | Eur. J. Biochem. 269 6195-6203 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03338.x The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains Yukiko Shimada1 Mikako Maruya2 Shintaro Iwashita3 and Yoshiko Ohno-Iwashita1 1 Biomembrane Research Group Tokyo Metropolitan Institute of Gerontology department of Cell Biology Tokyo Metropolitan Institute of Medical Science 3Mitsubishi Kagaku Institute of Life Sciences MITILS Machida Tokyo Japan There is much evidence to indicate that cholesterol forms lateral membrane microdomains rafts and to suggest their important role in cellular signaling. However no probe has been produced to analyze cholesterol behavior especially cholesterol movement in rafts in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts we prepared and characterized several truncated fragments of 0-toxin perfringolysin O a cholesterol-binding cytolysin whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C-terminal domain domain 4 of the toxin is the smallest functional unit that has the same cholesterol-binding activity as the full-size toxin with structural stability. Cell membrane-bound recombinant domain 4 was detected in the floating low-density fractions and was found to be cofractionated with the raft-associated protein Lck indicating that recombinant domain 4 also binds selectively to cholesterol-rich rafts. Furthermore an enhanced green fluorescent protein-domain 4 fusion protein stains membrane surfaces in a cholesterol-dependent manner in living cells. Therefore domain 4 of 0-toxin is an essential cholesterol-binding unit targeting to cholesterol in membrane rafts providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells. Keywords raft microdomain cholesterol BIAcore perfringolysin O. In recent years accumulating evidence has .
