Published online before print October 4, 2005
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* Department of Cell Biology in the Center of Anatomy and
Transplantationslabor, Klinik für Viszeral- und Transplantationschirurgie, Hannover Medical School, Germany; and
Department of Food Chemistry, University of Hamburg, Germany
1Correspondence: Department of Cell Biology in the Center of Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: rli{at}zellbiologie.mh-hannover.de
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Key Words: antigen presentation • membrane microdomain • detergent resistance • GEMs • DIGs
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Lipid rafts are membrane domains, which are enriched in cholesterol and sphingolipids and contain a specific subset of membrane proteins including glycophosphatidylinositol-anchored proteins (GPIs) [11
12
13
]. It is believed that the long and saturated acyl chains of sphingolipids pack with cholesterol and thus promote the formation of a liquid-ordered phase, whereas unsaturated glycerophospholipids favor a more fluidic state of cell membranes. Recently, evidence was provided for precursors of lipid rafts, which are 
5 nm in size, contain only one to four proteins, and persist less than a millisecond [14
, 15
]. After ligand- or antibody-mediated cross-linking, the precursors are thought to fuse and form larger and more permanent structures, which can be visualized as rafts by immunofluorescence techniques at the cell surface. They have been shown to serve as platforms for signaling and protein sorting [16
17
18
]. Operationally, lipid rafts can be defined as detergent-resistant membranes (DRMs) as a result of their partial resistance to solubilization by mild detergents [13
]. It is still a matter of controversy how closely DRMs resemble native lipid rafts, but it should be kept in mind that many of the proteins and lipids that associate with rafts after antibody-mediated cross-linking or ligand-induced oligomerization are found in DRMs. This suggests that the isolation of membrane domains with detergents may result in the formation of raft-like structures similar to those obtained after experimental or ligand-induced cross-linking at the cell surface. Over the last years, evidence had accumulated that there might be diverse types of lipid rafts and correspondingly, also different types of DRMs [19
20
21
22
23
24
25
]. It is interesting that some DRMs appear to be resistant only to weak detergents such as Lubrol WX, Brij 96, and Brij 98 but are solubilized by stronger detergents such as Triton X-100 (TX-100).
In a number of studies, the association of MHC II with TX-100-resistant membranes was examined; however, the results varied greatly. In many instances, only 1–5% of total MHC II was found in TX-100-resistant membranes [8
, 26
27
28
29
], whereas in other studies, a substantial amount of MHC II (more than 10%) was detected in TX-100-resistant membranes [5
, 6
, 10
, 29
, 30
]. In addition to TX-100, other detergents, such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) [31
], Brij 58 [32
], and Brij 98 [9
], have been used for the isolation of DRMs containing MHC II. With these detergents, a high fraction of MHC II (60%) was found to be associated with DRMs. Currently, it is not clear to what extent these differences were caused by variations in the isolation protocols rather than by biochemical properties of membranes containing MHC II and the invariant chain or by differences in cell lines and their signaling and/or activation status. For that reason, we compared isolation efficiencies and specificities using membrane preparations from mouse B-lymphoma cells, which were extracted with TX-100, CHAPS, or Brij 98. In addition, for every detergent, two variants of the DRM flotation protocol were tested. Our results demonstrate a large difference in efficiency and specificity of isolation of MHC II- and invariant chain-containing DRMs between the different protocols using TX-100, CHAPS, and Brij 98. This suggests that MHC II- and invariant chain-containing DRMs may share properties with DRMs occupied by the T cell receptor (TCR) complex [22
], prominin [20
], or the prion protein [19
, 25
].
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of DRMs
M12.C3.F6 B-lymphoma cells (3x107) were washed and resuspended in 1 ml 10 mM triethanolamine/acetic acid, 1 mM EDTA, and 250 mM sucrose, pH 7.4, supplemented with protease inhibitors (5.1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml E-64, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM iodoacetamide; HB-T+). In this buffer, the cells were homogenized by repeated passage through a 23-gauge needle, and a postnuclear supernatant was prepared (1000 g, 10 min, 4°C). After re-extraction of the nuclear pellet with 200 µl HB-T+, the combined supernatants were digested with 100 µg/ml DNase I for 1 h on ice. Postnuclear membranes were pelleted (40,000 rpm, 45 min, 4°C) in a TLA 45 rotor (Beckman Coulter, Fullerton, CA) and resuspended in 500 µl 20 mM 2-[N-morpholino]ethanesulfonic acid (MES) acid, 150 mM NaCl, 0.02% NaN3, and protease inhibitors (MES+ buffer) or in MES+ buffer containing 1% TX-100, 1% or 4% CHAPS, or 1% Brij 98 for extraction. After addition of DNase I (100 µg/ml), the postnuclear membranes were extracted for 1 h on ice. For extraction with Brij 98, a 5-min incubation at 37°C was done before the 1-h incubation step on ice. After extraction, all samples were mixed with 500 µl 90% sucrose containing the same concentration of detergent as in the extraction and transferred to an SW-41 tube. A 10-ml, 0–40% gradient of sucrose in MES+ buffer with or without detergent was layered on top of the sample, which was then centrifuged (38,000 rpm, 18.5 h, 4°C) in an SW41 rotor (Beckman Coulter). Eleven fractions were collected from the top, and the pellet was resuspended in 1 ml extraction buffer.
Gel electrophoresis, blotting procedures, and quantification
Standard sodium dodecyl sulfate gel electrophoresis was performed in mini gels containing 11% polyacrylamide. For Western blotting, primary antibodies were used as recommended by the manufacturer or at 1 µg/ml. CTB was used at 1 µg/ml and the anti-CTB antiserum at a 1:2000 dilution. Secondary reagents were diluted according to the recommendations of the manufacturers. For dot blots, 3 µl each gradient fraction was spotted onto a nitrocellulose membrane, dried, and incubated with antibodies as described above. All blots were developed with ECL reagent and exposed to film. Suitable exposures were scanned and quantified using the program NIH Image.
Lipid analysis
DRM fractions were pooled, diluted with 1:3 with MES buffer, and spun for 2 h at 100,000 g at 4°C. Pellets were resuspended in 1.8 ml 0.9% and NaCl, 0.05% HCl ("salty acid") and transferred to glass tubes, and 2 ml methanol and 2 ml chloroform were added sequentially. After shaking, samples were incubated for 10 min on ice and spun for 5 min at 300 g. The upper phase was removed, and 2 ml methanol and 1.8 ml salty acid were added to the lower phase. Samples were again incubated and spun as above, and the upper phase together with the interphase were removed this time. Chloroform (1 ml) was added to the lower phase, and speed-vac centrifugation was performed. Dried pellets were resuspended in 20 µl chloroform, and 3 µl each sample was loaded onto HPTLC sheets. Lipids were resolved by using a two-solvent system. Samples were first run to the halfway point of the plate in a hydrophilic solvent (50:20:10:10:5 chloroform/acetone/acetic acid/methanol/water) and air-dried and finally, ran in a hydrophobic solvent (50:20 hexane/ethyl acetate). Lipids were visualized with the fluorescent dye primulin (0.05% in an 80:10 mixture of acetone and water, respectively), photographed with a digital camera, and quantified using NIH Image. The relative abundance of the lipids was calculated under the assumption that the molar primulin adsorption is similar among the lipids we analyzed.
Antigen presentation assays
As antigen-presenting cells (APCs), we used the B-lymphoma line M12.C3.F6, which had been cultured in the presence or the absence (controls) of 2 µM HEL for 24 h. After washing, 100 µl B cells (105cells/ml) were incubated with 50 µl 3A9 T hybridoma cells specific for the HEL peptide 48–62 (2x106 cells/ml) for 24 h at 37°C. Alternatively, after incubation with HEL, 3 x 107 APCs were processed for DRM isolation using MES+ buffer containing 1% of TX-100 or Brij 98 for extraction and 1% of each detergent in the gradient. The DRM fractions were identified by dot-blotting against GM1, pooled, diluted 1:3 with MES+ buffer, and pelleted by centrifugation at 90,000 rpm (TLA 100.4 Beckman rotor) for 1 h at 4°C. After washing two times in phosphate-buffered saline (PBS), the pelleted membranes were resuspended in 500 µl RPMI-1640 medium with all supplements (see above). The 3A9 cells were then incubated with 30 µl resuspended DRMs (approximately 1x106 cell equivalents, assuming a 50–60% isolation efficiency) for 24 h at 37°C in a cell-culture incubator. Supernatants were collected the following day and frozen at –20°C to kill any remaining cells. After thawing, 50 µl supernatants were incubated with 50 µl CTLL-2 cells (105 cells/ml) at 37°C in a cell-culture incubator. After 24 h, 20 µl solution of 0.05 µCi/µl [3H] thymidine was added to each sample, and the culture was continued for an additional 24 h. Finally, cells were harvested on glass fiber mats (Inotech, Dottikon, Switzerland), and the incorporated [3H] thymidine was measured in a Wallac-Trilux microbeta reader 1450.
Enzymatic reactions
For detection of alkaline phosphatase, 50 µl substrate containing 20 mM p-nitrophenylphosphate, 2 M diethanolamine, and 2.5 mM MgCl2, pH 9.8, was added to a 50-µl sample in a microtiter plate and incubated for 2–4 h at 37°C. Color development was read at 404 nm in an enzyme-linked immunosorbent assay reader.
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30% of the GPI-anchored protein AP were found to float under these conditions, together with a low amount of MHC II. The TfnRC, a protein not considered to be a raft component at the plasma membrane [39
], did not float appreciably under this condition. The high-mannose ER form and the mature form of invariant chain and the nonraft markers lamp-1 [9
, 40
] and B220 [6
] were detected in DRM fractions, at least to a similar extent as MHC II. Taking into consideration that ER proteins are not associated with lipid rafts [41
], the latter result made it difficult to determine whether the small amount of MHC II in the gradients lacking the detergent reflected genuine or only nonspecific DRM association. To answer this question, we tried to increase the selectivity by including the detergent in the gradient (Fig. 1B)
. Under these more stringent conditions, only the raft markers GM1 and AP were floating (albeit to a slightly lower extent), and the nonraft markers lamp-1 and B220 along with TfnRc, MHC II, and the invariant chain were found exclusively in the soluble fractions. Thus, the presence of detergent in the gradient prevented the low, nonspecific flotation of lamp-1, B220, and the immature ER form of the invariant chain as well as the flotation of small amounts of MHC II and the mature invariant chain. This suggests that the latter two proteins did not float specifically into the gradient when the detergent was absent. We speculate that the presence of detergent in the gradient may prevent reformation of membrane vesicles caused by the dilution of the detergent during flotation. Alternatively, the extended exposure to the detergent during centrifugation may lead to a solubilization of membranes, which resist the detergent for only a limited time. It is also possible that weakly DRM-associated proteins dissociate from the membranes under nonequilibrium conditions in the gradient. To investigate whether membrane lipids were affected by the presence of the detergent in the gradient, we analyzed the lipid composition of DRMs isolated in the presence or absence of 1% TX-100 from the gradient (Fig. 2
). DRMs isolated in the presence of 1% TX-100 showed a higher enrichment in cholesterol (35% of the primulin signal for these DRMs) and Sm/PI (20%) compared with DRMs retrieved in the absence of detergent (cholesterol: 22%; Sm/PI: 12%) or total postnuclear membranes (cholesterol: 18%; Sm/PI: 10%). The enrichment of Sm/PI and cholesterol demonstrates that the presence of TX-100 in the gradient indeed influenced the membrane lipid composition. Thus, it is possible that the detergent in the gradients did not induce the dissociation of proteins from DRMs but instead, dissolved the membranes containing these proteins.
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Figure 1. Flotation of TX-100-extracted, postnuclear membranes on linear 0–40% sucrose gradients in the absence (A) or the presence (B) of 1% TX-100 throughout the gradient. Postnuclear membranes were prepared from M12.C3.F6 mouse B-lymphoma cells, extracted in MES+ buffer containing 1% TX-100, and floated in 0–40% sucrose gradients with (B) or without (A) detergent. Individual fractions were analyzed by Western blotting with the mAb 10.2.16 (MHC II), In-1 (invariant chain), H86.4 [transferrin receptor (TfnRc)], 1D4B (lamp-1), or cholera toxin B-subunit (GM1) or by dot blotting with the mAb RA3-6B2 (B220). The raft marker alkaline phosphatase (AP) was quantified by enzyme assay. Note that in A, only little MHC II floated into the gradient along with a similar amount of lamp-1, B220, and TfnRc. Also note that no flotation of MHC II, invariant chain, or nonraft markers was found when TX-100 was present in the gradient (B). Iimat., Mature invariant chain; Iiimm., immature invariant chain (ER form); a.u., arbitrary units; OD, optical density; p, pellet.
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Figure 2. Lipid composition of TX-100-resistant membranes isolated from gradients with or without detergent in comparison with total postnuclear membranes. Lipids were extracted from membrane pellets as described in Materials and Methods, separated by HPTLC, stained with primulin, and quantified with NIH Image. The relative abundance of lipids was calculated under the assumption that all lipids showed a similar molar primulin adsorption. Sm and PI were not separated sufficiently in our solvent system and thus, were quantified together. The data represent the mean of three independent experiments, and error bars indicate the SD. Note that DRMs isolated in the continued presence of TX-100 contain the highest relative amount of cholesterol and Sm/PI but the lowest relative amount of PC and PE.
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Figure 3. Flotation of postnuclear membranes extracted with 1% CHAPS on linear 0–40% sucrose gradients in the absence (A) or the presence (B) of 1% CHAPS in the gradient. Postnuclear membranes from M12.C3.F6 were extracted with 1% CHAPS for 1 h on ice and overlayed with 0–40% linear sucrose gradients in the presence/absence of 1% CHAPS. Fractions were analyzed as detailed in the legend to Figure 1
. Note that in A, the raft markers GM1 and AP reached peak levels in fractions 5–7, whereas the other proteins partially floated to a distinct position (fractions 7–8).
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Figure 4. Flotation of postnuclear membranes extracted with 4% CHAPS in the absence (A) or the presence (B) of the detergent in the gradient. Postnuclear membranes from M12.C3.F6 cells were extracted with 4% CHAPS for 1 h on ice and overlayed with 0–40% linear sucrose gradients with or without 4% CHAPS. Fractions were again analyzed as detailed in the legend to Figure 1
. Note that a higher degree of mature invariant chain was found in the gradient at a position to which AP was also distributed.
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30% for Brij 98 DRMs vs. 18% for total postnuclear membranes) and a markedly lower content of PC (31% for Brij 98 DRMs vs. 47% for total postnuclear membranes). This is similar to TX-100-resistant membranes, which were prepared in the presence of detergent in the gradient (Fig. 2)
. However, in the latter case, the relative abundance of Sm/PI was also significantly higher than in postnuclear membranes (Fig. 2)
, whereas with Brij 98 DRMs, the minor increase over the value for postnuclear membranes remained insignificant. Taken together, we conclude that both Brij 98 protocols allow efficient and specific isolation of DRMs containing MHC II and the mature invariant chain. These DRMs are similar in their lipid composition to the TX-100-resistant membranes prepared in the presence of the detergent in the gradient.
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Figure 5. Flotation of postnuclear membranes extracted with 1% Brij 98 in the absence (A) or the presence (B) of the detergent in 0–40% linear sucrose gradients. Postnuclear membranes from M12.C3.F6 cells were extracted with 1% Brij 98 for 5 min at 37°C followed by 1 h on ice. Samples were overlayed with 0–40% linear sucrose gradients with or without 1% Brij 98. Fractions were analyzed as detailed in the legend to Figure 1
. Note that a sizeable fraction of MHC II and almost all the mature invariant chain floated to DRM fractions marked by GM1 and AP, and only a little immature invariant chain and no lamp-1 were found in the gradients.
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Figure 6. Lipid composition of Brij 98-resistant membranes isolated from gradients with or without detergent in comparison with total postnuclear membranes. Lipids were extracted from membrane pellets as described in Materials and Methods, separated by HPTLC, stained with primulin, and quantified with NIH Image. The relative abundance of lipids per sample was calculated under the assumption that all lipids showed a similar molar primulin adsorption. Sm and PI were quantified together, as already explained in the legend for Figure 2
. The data represent the mean of three independent experiments, and error bars indicate the SD. Note the high abundance of cholesterol and the lower level of PC in DRMs compared with the postnuclear membranes.
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Figure 7. Antigen presentation by DRMs. M12.C3.F6 cells were cultured with 2 µM HEL for 24 h, and aliquots (1x104 cells) were cocultured with 3A9 T cells specific for HEL 48–62 (1x105 cells). Alternatively, DRMs were isolated from HEL-pulsed cells in the presence of 1% Brij 98 or 1% TX-100 throughout the procedure. For presentation, PBS-washed DRMs (approximately 1x106 cell equivalents) were cocultured with 1 x 105 3A9 cells. The IL-2 production by 3A9 cells was quantified with the IL-2-dependent cell line CTLL-2. Because of considerable variations in the overall [3H] thymidine incorporation between individual experiments (compare A with B), data from one out of two experiments are shown, each performed in triplicates. Error bars denote the SD of the triplicates. (A) Antigen presentation by Brij 98 DRMs prepared from M12.C3.F6 cells pulsed with 2 µM HEL. The background proliferation of CTLL-2 cells in this experiment was 390 ± 110 counts per minute (cpm). (B) Antigen presentation by TX-100 DRMs prepared from M12.C3.F6 cells pulsed with 2 µM HEL. The background proliferation of CTLL-2 cells in this experiment was 6706 ± 288 cpm. Note that TX-100 DRMs from HEL-pulsed cells did not stimulate 3A9 T cells, whereas Brij 98 DRMs from HEL-pulsed M12.C3.F6 cells did. Positive control (APCs+HEL): M12.C3.F6 cells pulsed with 2 µM HEL; mixing experiment (APCs+HEL+DRMs): M12.C3.F6 cells (1x104) pulsed with 2 µM HEL were mixed with washed TX-100 DRMs prepared from untreated M12.C3.F6 cells.
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In our hands, the TX-100 DRM isolation protocol using flotation in the absence of detergent yielded barely more than background levels of MHC II and mature invariant chain in DRM fractions (each <<5% of total). Our observations are similar to results from a number of studies about the raft association of MHC II, which used extraction with 0.5–1.0% TX-100 and flotation in detergent-free 5/30% sucrose step gradients as a readout [5 , 8 , 26 27 28 29 ]. In these studies, values for the MHC II-DRM association ranging from 1% to 5% have been reported for unstimulated B cells, dendritic cells (DCs), and monocytes with usually undetectable levels of background flotation. In some instances, however, a substantially higher DRM association of MHC II using similar TX-100-based isolation procedures has been shown [6 , 8 , 10 , 29 , 30 ]. These higher values may have been obtained for different reasons (for a discussion of the results reported in ref. [30 ], see ref. [9 ]). In some studies, a higher DRM association of MHC II was reported after surface cross-linking with mAb against MHC II and appropriate "secondary" antibodies, a regimen described to induce clustering to microscopically visible lipid rafts at the cell surface [39 ]. This was also shown to lead to an incremental increase in DRM association of MHC II [5 , 8 , 27 ]. Another way to increase the association of MHC II molecules with TX-100-resistant membranes appears to be the maturation of DCs [29 ] or the loading of MHC II with a high-affinity peptide [10 ]. As mature DCs present an edited set of peptides on MHC II, which differs from the one in immature DCs [44 ], the underlying molecular cause for both observations may be identical. This interpretation is consistent with results from an earlier study about the association of MHC II with Brij 98-resistant membranes, demonstrating that MHC II-bound peptides modulate the recruitment of their carrier molecule to DRMs [9 ]. This effect may also explain differences between some B cell lymphoma lines, where in some instances, a slightly higher association of MHC II with TX-100-resistant membranes was found (10–20% of total compared with 1–5% found for most unstimulated APCs) [6 , 10 ]. It is interesting that none of these studies used TX-100 in the sucrose gradients to rule out artifactual flotation. As shown in Figure 1B , we did not observe any flotation of MHC II in the presence of TX-100 in the gradient, although genuine raft markers still were afloat. This and the altered lipid composition of DRMs isolated with TX-100 in the gradient (Fig. 2) suggest that membrane domains containing MHC II (and mature invariant chain) differ from those containing only conventional raft markers.
A similar conclusion was reached when we analyzed the association of MHC II and the mature invariant chain with CHAPS-resistant membranes in comparison with conventional lipid raft markers. We were unable to achieve significant, specific flotation of MHC II (and the mature invariant chain) using this detergent. This is in line with data by others, who also have noted that total MHC II from a B cell line does not distribute to DRMs after CHAPS extraction [45 ]. By contrast, MHC II isolated from multivesicular endosomes and exosomes efficiently partitioned to CHAPS-resistant membrane fractions along with tetraspanins [45 ]. In fact, a minor subpopulation of MHC II (<5–10% of total MHC II in B cells) molecules has been shown to associate with tetraspanin networks [26 ], and its low abundance in cellular membranes may prevent its unequivocal detection above background flotation.
In contrast to TX-100 and CHAPS, the presence or absence of Brij 98 from the gradients produced only little difference in the flotation of raft markers, MHC II, and the mature invariant chain. This result was confirmed by lipid analysis, which yielded almost identical values for both DRM isolation protocols. It is interesting that the lipid compositions of DRMs prepared with the Brij 98 protocols or the stringent TX-100 protocol (with detergent in the gradient) were, on one hand, quite similar to each, and conversely, they were significantly different from postnuclear membranes. By contrast, the lipid composition of DRMs prepared with the conventional TX-100 protocol, which omits the detergent from the gradient, showed more similarity to postnuclear membranes, in line with results reported by Simons and colleagues [38 ]. These results suggest that in our hands, Brij 98 procedures and the stringent TX-100 protocol yielded DRMs with lower contamination of nonraft membranes than the commonly used TX-100 protocol without detergent in the gradient [38 , 41 ]. Despite these similarities between the DRMs prepared with the stringent TX-100 protocol and the Brij 98-based procedures, there were also differences: Proteins such as MHC II, the mature invariant chain, and endosomal TfnRc [9 ] were only present in Brij 98 DRMs, whereas the relative abundance of SM/PI was lower in these DRMs. These data are compatible with the interpretation that Brij 98 DRMs selectively contain additional membrane components in comparison with DRMs isolated by the stringent TX-100 protocol. This view is supported by data about the ability of Brij 98 DRMs to activate T cells and the inability of TX-100 DRMs to do so, which apparently did not contain sufficient MHC II-peptide complexes to drive antigen presentation. To our knowledge, this is the first measurement of T cell activation by isolated DRMs reported in the literature.
To date, there is a small number of untypical raft proteins, which show a similar type of behavior to what we described here, such as the TCR complex [22 ], the microvillus protein prominin [20 ], or the prion protein [19 , 25 ]. These proteins remain mostly soluble when extracted with 1% TX-100 but resist extraction by milder detergents such as Brij 96, Lubrol WX, or Brij 98. For these three proteins, evidence has been provided that differential detergent sensitivity correlates with localization to distinct and separate membrane patches at the cell surface [19 , 20 , 25 ], with differential enrichment in immunoisolated DRMs [19 , 22 , 25 ] or with a partial but direct biophysical separation on linear flotation gradients [20 ]. All of this suggests that the TCR complex, prominin, and the prion protein indeed localize to membrane domains distinct and physically separated from conventional lipid rafts. The changes we observed in lipid composition upon inclusion of TX-100 in the flotation gradients, the elimination of floating MHC II and the mature invariant chain under this condition, and the efficient and specific flotation of these two proteins in Brij 98 show that MHC II and the mature invariant chain follow a similar pattern and thus, strongly suggest that they also localize to membrane domains distinct from conventional lipid rafts. It is interesting in this respect that MHC II (this work) and the TCR [22 , 46 , 47 ] appear to prefer a closely related or identical membrane environment. This may result in a similar packing geometry of these proteins, which may favor the corecruitment of this receptor-ligand pair into the dense center of mature, immunological synapses. At present, however, our data do not rule out the possibility that MHC II, the mature invariant chain, and other membrane components are associated with the edges of a TX-100-resistant "core raft" [48 ] and thus, do not form a separate entity. Immunoisolation experiments should help to distinguish between these two possibilities in the future.
Received April 12, 2005; revised June 23, 2005; accepted July 15, 2005.
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