Ii3, thus abolishing the binding affinity of Ii3 for further ab heterodimers. Discussion Shortly after biosynthesis in the ER, MHCII a and b chains form transient aggregates [33]. Expression of Ii enables the release of a and b chains from such aggregates and facilitates correct assembly of the MHCII subunits [34]. When assembly is accomplished, the MHCII-Ii complex exits the ER. The three- dimensional structure of the MHCII-Ii complex has not yet been resolved by crystallography or nuclear magnetic resonance, and thus its composition is still not fully clarified. In the initial report by Roche et al. [12], the authors concluded from crosslinking experiments that a band of lower mobility than 220 kDa molecular weight marker represented the crosslinked DR-Ii complex, and they hypothesized that the MHCII-Ii complex is composed of nine subunits, an Ii trimer and three DR heterodimers, with a total molecular weight of 260 kDa [35]. In agreement with the original observation by Roche and collabo- rators, we find that DR indeed crosslinks into high molecular weight complexes, but we also find that such complexes are of a very broad size range and that besides Ii (and where applicable, MHCII) they may contain other cellular components. Our results indicate that crosslinking experiments alone are not sufficient to accurately predict the size of the MHCII-Ii complex. In this study, we have investigated the stoichiometry of MHCII- Ii complexes by using epitope-tagged subunits of MHCII and assessing their co-isolation with endogenous components. In contrast to the work cited above, we find strong evidence for a pentameric complex consisting of an Ii trimer and one dimer of MHCII. Several lines of evidence from the literature support our findings that Ii forms distinct complexes with the MHCII isotypes. Initial studies in mice were performed with antisera specific for H-2A or H- 2E isotypes of Ia (mouse MHCII) [15]. Such locus-specific antisera separately isolated metabolically labeled H-2A and H-2E isotypes bound to Ii, but did not coprecipitate H-2A with H-2E or vice versa [36]. The co-isolation of H-2A and H-2E that did occur with some monoclonal antibodies was found to come from cross-reactivity of the monoclonal antibodies to the two isotypes [37]. Likewise, in humans, the cross-reactivity of many anti-HLA-class II monoclonal antibodies with several of the three isotypes may explain why distinct complexes of DP, DQ, and DR with Ii have not been clearly demonstrated to date. We show here that DP or DQ immunoprecipitates do not contain DR subunits. This result suggests that the Ii trimer only associates with one of the DP, DQ, or DR isotype heterodimers. The direct determination of size of the Ii-MHCII complex is naturally difficult because of the highly elongated nature of Ii, which leads to an apparent size of twice its molecular mass in gel filtration and light scattering [2], and because of the association of detergent with the protein, which might lead to an artefactual increase in the hydrodynamic radius [38]. Thus, in a combined gel filtration/sedimentation velocity ultracentrifugation experiment, a mass of 390 kDa was calculated for the DR-Ii complex [12], with the deviation of this value from the calculated mass of the nonameric Ii3(ab)3 complex (260 kD, see above) assigned to detergent. In our experiments by separation of cell lysates in a sucrose gradient and comparison to protein standards, we estimate a molecular weight of about 130 to 160 kDa for the DR-Ii complex. Here, the sedimentation properties of the protein are not entirely determined by its association with detergent micelles, since we can very well separate small DRab from large DRabIi complexes (Figures 1, 2 and 3). HLA-Class II-Invariant Chain Complexes To investigate whether the use of a different detergent, Triton X-100, was responsible for our differing size estimate, we examined two additional detergents, n-octylglycoside and digito- nin for separation of MHCII complexes in a sucrose gradient. In our hands, the sucrose gradient with either of these detergents did not separate peptide loaded MHCII molecules from MHCII-Ii complexes (data not shown). To show that the use of Triton X-100 did not lead to dissociation of a larger complex, we then demonstrated that Ii-DR complexes from digitonin-lysed cells are indeed stable in Triton X-100 (Fig. S1F). We therefore conclude that Triton X-100, which can be used to separate MHCII-Ii complexes from Ii free MHCII heterodimers, allows the isolation of intact and complete Ii-DR complexes. Nuclear magnetic resonance studies of recombinant soluble Ii have shown that the MHCII binding site is contained in a disordered region of Ii [39] that acquires an ordered conformation upon MHCII binding [19]. In vitro assembly of titrated soluble Ii with constant amounts of soluble DR (truncated just before the transmembrane domain) showed additional bands in a non- denaturing gel at decreasing Ii concentrations. This result was taken to suggest that Ii oligomers associate with several pairs of MHCII subunits [19]. Our data, in contrast indicate that only one full-length MHCII heterodimer is contained in the MHCII-Ii oligomer, resulting in a pentameric Ii3ab complex. Our molecular model of the complex (Fig. 9) is not per se evidence for a pentameric complex, but it conclusively demon- strates that the pentameric model is compatible with all known structural data, and it suggests a possible mechanism for unique binding of a MHCII heterodimer to Ii3. We postulate that upon binding of a single MHCII heterodimer to one unstructured CLIP sequence, the triple-helical core domain of Ii3 changes its conformation, which imposes a steric constraint onto the entire complex, discouraging binding of further MHCII heterodimers. Interestingly, the murine MHC class I molecule H-Kb, which is structurally related to MHCII molecules, was suggested to adopt a supine (lying down) orientation on the membrane, with the peptide binding site close to the membrane (while remaining accessible to the T cell receptor and the CD8 coreceptor [40]). If an MHCII ab heterodimer adopted a similar conformation, then Ii would need to bent over extremely strong to interact with them, which would presumably prevent the binding of a second and third MHCII ab heterodimer. Conclusive evidence to the nature and degree of the conformational change of Ii3 upon MHCII ab binding will probably only be obtained upon the resolution of the three- dimensional structure of the complex. Why would Ii itself form a trimer if it does not bind three MHCII heterodimers? We can offer several hypothetical expla- nations. First, Ii contains several topogenic sequences in its cytosolic tail that are responsible for internalization from the cell surface and for the targeting of the complex to endosomes [41]. Trimerization of Ii is required for the efficient targeting of MHCII-Ii to endosomal compartments [42–43], suggesting that the trimeric character allows Ii to fulfil its function in targeting MHCII to endosomes more efficiently. The trimeric tail could have a similar function in ER-to-Golgi transport, where COPII coat of transport vesicles is known to recognize oligomeric cytosolic tails of proteins, and/or in other intracellular steps [44–45]. Second, Ii (CD74) plays a role as a cell surface receptor or coreceptor for a number of proteins, most prominently for the macrophage inhibitory factor (MIF). Since MIF itself is a trimer with rotational symmetry, a trimerization of Ii might be required to bind MIF [27], [46]. A pentameric MHCII- Ii complex has some distinct advantages for the process of MHCII antigen presentation. First, one would PLoS ONE | www.plosone.org 10 February 2011 | Volume 6 | Issue 2 | e17257