Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.
The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with beta-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.
Hayashi
Masami
M
Cellular Signaling Group, Research Team for Functional Genomics, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan.
Shimada
Yukiko
Y
Inomata
Mitsushi
M
Ohno-Iwashita
Yoshiko
Y
D016475:3T3 Cells; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol