complexes, and that Ii-DR complexes inside cells contain only one DRab heterodimer. An analogous experiment with DRb chain showed no co- isolation of endogenous DRb with DRbV5, confirming the observation with DRa (see Text S1, supporting information, Fig. S2A). In addition, this experiment was repeated with the detergent digitonin (Fig. S2B). Again, endogenous DRb was not co-isolated with DRb-V5. To exclude the possibility that the observed pentameric complexes were an artefact of transfection, and to verify that under non-transfected conditions DR and DQ segregate into individual complexes with Ii, we employed the mAb 1a3, which reacts with DQ but does not crossreact with DR. We lysed untransfected Raji cells and separated the lysates on sucrose gadients as before. In these gradients, the pooled fractions 7/8 contain peptide loaded MHCII heterodimers (e.g. DQab and DRab heterodimers), and fractions 14/15 harbour MHCII-Ii complexes (see Fig. 1 to 3). Both pools of fractions were immunoprecipitated for DQ (Fig. 7, lanes 1 and 2) and for DR (lanes 3 and 4) and subsequently immunoblotted for DRa (upper panels) or for DQb or DRb chains (lower panels). Immunopre- cipitation of DQ from fractions 7/8 (lane 1) shows no reaction with the DRa mAb (upper panel), confirming that mAb 1a3 does not crossreact to DR. Staining of DQb (lower panel, lane 1) indicates that 1a3 immunoprecipitates DQ. When fractions 14/15 were immunoprecipitated for DQ, the DQb chain was detected by western blotting (lane 2, lower panel). Strikingly, however, no DRa was coprecipitated with DQ from the gradient fractions containing MHCII-Ii complexes (lane 2, upper panel). As a control, immunoprecipitation of DR shows that DRa (upper panel) and DRb (lower panel) are indeed present in both pools. This result suggests that in untransfected cells, complexes of DQ with Ii do not contain DR. Figure 7. Isolation of DQ-Ii complexes from sucrose gradient fractions. Sucrose gradient fractions of Raji cell lysates were subjected to immunoprecipitation with mAb 1a3 against DQ and with DR mAb ISCR3. Fractions 7 and 8 or 14 and 15 were pooled and immunopre- cipitated. DQ (lanes 1 and 2) or DR (lanes 3 and 4) were isolated, and western blotted for DRa (upper panel, mAb TAL-1B5) or for DQb (lower panel, lanes 1 and 2) and for DRb (lanes 3 and 4) with mAb LGII-612.14. The position of DRa, of DQb or of DRb and bands corresponding to immunoglobulin heavy (H) and light (L) chains are labeled by arrows. doi:10.1371/journal.pone.0017257.g007 HLA-Class II-Invariant Chain Complexes Subunit composition of DR-Ii oligomers Finally, we wished to analyze the formation of the complexes of DR and Ii, which occurs soon after biosynthesis in the ER. DRbV5-transfected MelJuSo cells were pulse labeled with 35S- methionine for 10 min. (At this short time point, the majority of labeled MHCII and Ii chains localize to the ER). Cells were then lysed, and DRbV5 was immunoprecipitated with anti-V5 monoclonal antibody or with the conformation-dependent mono- clonal antibody I251SB against DRab. To assess the mobility of the individual polypeptide chains, Ii, DRb, DRa and DRbV5 were immunoprecipitated from lysates of transfected COS-7 cells (Fig. 8, lanes 1 to 4). Immunoprecipitation of DRbV5 from lysates of the transfected cells revealed two bands (Fig. 8, lane 5), but no band for endogenous DRb, suggesting that as in the steady-state experiment (Fig. S2), the transfected DRbV5 was not associated with endogenous DRb via Ii The lower band in lane 5 corresponds to the mobility of Ii, and the upper band contains both DRa and DRbV5, as shown by the separation of immunoprecipitates of lanes 4 and 5 on a long gel (right panel). Lane 5 also indicates that DRbV5 is not overexpressed in comparison to endogenous DRa. The results of this experiment suggest that the ER form of the DRabIi complex contains only one DRb chain and not several. To rule out that the labeling time (ten minutes), was to short to allow for the formation of a larger complex, we pulse labeled DRbV5 expressing cells for 10 minutes with 35S methionine chased for 40 minutes with unlabeled methionine, and immuno- precipitated DRbV5 (lane 11). As in lane 5, two bands containing DRbV5 plus DRa and Ii were separated whereas endogenous DRb could not be detected. Thus, the complex of DR and Ii contains only one DRb chain. A revised model for MHCII-Ii oligomers Finally, we attempted to reconcile our data with the existing knowledge of the three-dimensional structure of MHCII and Ii using a computational model. At first, we considered the structure of Ii3. The primary sequence of Ii shows a repeat of leucine pairs, starting at the cytosolic tail at Leu-7 of the Ii 33 kDa form of the polypeptide. The leucine residues appear in a distance of seven amino acids and this motif is repeated five times through the membrane spanning region towards the luminal part up to residue 81, suggesting an extensive interhelical interaction such as a leucine zipper. Since the three Ii chains are known to interact within the membrane [32], we postulate that the trimeric domain of Ii extends from residues Leu-7 to Leu-81. Figure 9 shows a structural model in which the amino terminus of Ii shows a coiled structure composed of three Ii subunits, followed by a non-ordered sequence between amino acid 82 and the C-terminal trimeric domain. This loop contains the CLIP sequence, which fills the cleft of the MHCII heterodimer [6]. In the model, the binding of the CLIP sequence to the MHCII heterodimer is sterically possible in the complex if one assumes a slightly kinked arrangement of the MHCII molecule with respect to the membrane. The other two Ii chains are indicated in the same conformation as that associated with an MHCII heterodimer. It has been shown that the CLIP containing segment is disordered in the absence of a bound MHCII heterodimer [19]. Hence, the segment structure of the two unbound Ii-chains in the model represents one among several possible conformations. According to the structural model, binding of one MHCII molecule would not sterically exclude the possibility of two other Ii chains to adopt a near-bound conformation of the CLIP segment. However, consistent with a pentameric structure, it is possible that binding of one MHCII heterodimer restricts the conformational freedom of the Ii trimer by introducing a kink in PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e17257