Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution.
Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.
Korzeniowski
Marek
M
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
Kwiatkowska
Katarzyna
K
Sobota
Andrzej
A
D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000737:chemistry*; D004351:Drug Resistance; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D017830:Octoxynol / Q000737:chemistry*; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D020298:U937 Cells
D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D011948:Receptors, Antigen, T-Cell; D017452:Receptors, IgG; D017830:Octoxynol