Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

被引:182
作者
Waheed, AA
Shimada, Y
Heijnen, HFG
Nakamura, M
Inomata, M
Hayashi, M
Iwashita, S
Slot, JW
Ohno-Iwashita, Y
机构
[1] Tokyo Metropolitan Inst Gerontol, Dept Prot Biochem, Itabashi Ku, Tokyo 1730015, Japan
[2] Univ Utrecht, Ctr Med, Dept Hematol, Div Thrombosis & Hemostasis, NL-3508 GA Utrecht, Netherlands
[3] Univ Utrecht, Ctr Med, Dept Cell Biol, NL-3508 GA Utrecht, Netherlands
[4] Univ Utrecht, Ctr Med, Biomembrane Inst, NL-3508 GA Utrecht, Netherlands
[5] Mitsubishi Kasei Inst Life Sci, Tokyo 1948511, Japan
关键词
D O I
10.1073/pnas.091090798
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC theta) of perfringolysin O (theta -toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC theta on a sucrose gradient. BC theta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC theta binds to a subpopulation of vesicles in FLDF, Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC theta binding to FLDF. The staining patterns of BC theta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC theta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC theta binding does not cause any damage to cell membranes, indicating that BC theta is a useful probe for the detection of membrane rafts in living cells.
引用
收藏
页码:4926 / 4931
页数:6
相关论文
共 33 条
[1]   Fluorometric method for the enzymatic determination of cholesterol [J].
Amundson, DM ;
Zhou, MJ .
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, 1999, 38 (01) :43-52
[2]   The caveolae membrane system [J].
Anderson, RGW .
ANNUAL REVIEW OF BIOCHEMISTRY, 1998, 67 :199-225
[3]   Thiol-activated cytolysins: structure, function and role in pathogenesis [J].
Billington, SJ ;
Jost, BH ;
Songer, JG .
FEMS MICROBIOLOGY LETTERS, 2000, 182 (02) :197-205
[4]   Structure and function of sphingolipid- and cholesterol-rich membrane rafts [J].
Brown, DA ;
London, E .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (23) :17221-17224
[5]   Functions of lipid rafts in biological membranes [J].
Brown, DA ;
London, E .
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 1998, 14 :111-136
[6]   SORTING OF GPI-ANCHORED PROTEINS TO GLYCOLIPID-ENRICHED MEMBRANE SUBDOMAINS DURING TRANSPORT TO THE APICAL CELL-SURFACE [J].
BROWN, DA ;
ROSE, JK .
CELL, 1992, 68 (03) :533-544
[7]   Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36 [J].
Dorahy, DJ ;
Lincz, LF ;
Meldrum, CJ ;
Burns, GF .
BIOCHEMICAL JOURNAL, 1996, 319 :67-72
[8]   Active Lyn protein tyrosine kinase is selectively enriched within membrane microdomains of resting platelets [J].
Dorahy, DJ ;
Burns, GF .
BIOCHEMICAL JOURNAL, 1998, 333 :373-379
[9]   Landing on lipid rafts [J].
Fivaz, M ;
Abrami, L ;
van der Goot, FG .
TRENDS IN CELL BIOLOGY, 1999, 9 (06) :212-213
[10]   Microdomains of GPI-anchored proteins in living cells revealed by crosslinking [J].
Friedrichson, T ;
Kurzchalia, TV .
NATURE, 1998, 394 (6695) :802-805