Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction

We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BC theta. The BC theta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximate to 1.0) than the BC theta-unbound population in raft fractions (approximate to 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BC theta-bound membranes. On the other hand, CD3 epsilon, CD3 zeta and Zap70 are localized in the BC theta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3 epsilon, CD3 zeta and Zap70 are recruited to the BC theta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BC theta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.