assume that due to the unordered nature of the MHCII- binding domains of Ii, a pentamer would be more sensitive to proteolytic digestion in the endosome; two of the three Ii molecules would be rapidly cleaved, leading to the breakup of the Ii3 trimer. In a nonamer, the protease-resistent MHCII heterodimers would shield Ii from degradation in endocytic compartments. Second, in a pentameric complex, ER quality control of MHCII would probably be faster and more efficient. Usually, two alleles each of the a and b chains of DP and DQ isotypes are expressed (DRa is nonpolymorphic). From the numerous combinations of DP, DQ and DR a and b chains that are present in the ER, only a few can form matching heterodimers. The quality control in the ER permits exit of MHCII-Ii oligomers only after assembly of the matching MHCII chains, while mismatched ab heterodimers do not exit the ER [47]. The assembly of these matched heterodimers takes place on Ii3 [48]. If a nonameric complex was formed in the ER, three matched dimers would have to be found for the complex to leave the ER [49], whereas association of the Ii trimer with only one ab heterodimer would allow the immediate exit of a MHCII molecule from the ER, as soon as it is properly assembled. Substantial work is required to explore the cell biological consequences of our findings. Materials and Methods Cells, transfection, and biosynthetic labelling MelJuSo is a human melanoma cell line that expresses large amounts of HLA class II molecules and Ii. Monocyte-derived DCs were a kind gift of Dr. S. Koch, Determatology Department, Bonn. MelJuSo cells, Raji cells and COS-7 cells were cultured in DMEM containing 10% FCS. Cells were transfected with JetPEI (QBiogen, Heidelberg, Germany) according to instructions of the supplier. Briefly, 1 to 2 mg DNA were mixed with 2.5 to 5 ml JetPEI in 100 ml medium, added to 56105 cells, and subsequently the cells were cultured for three days. For labeling, 26106 cells were suspended in 200 ml RPMI medium without methionine and 100 mCi of 35S methionine were added. After a pulse of 10 minutes, the cells were pelleted by centrifugation and washed with cold PBS. The pulse labeled cells were chased with medium containing non-radioactive methionine. Immunoprecipitation, western blotting, and immunofluorescence microscopy Cells were lysed with 1% NP40 or with 1% Triton X-100 in Tris-buffered saline pH 7.4 containing protease inhibitors (Com- plete, Roche Diagnostics, Mannheim Germany). DNA and debris were removed by high speed centrifugation. Pre-absorption of the lysate was conducted with CL4B-Sepharose (Amersham, Freiburg, Germany). Immunoprecipitates were isolated with protein A Sepharose, separated by SDS-PAGE, and blotted onto Immobilon P membrane (Millipore, Schwalbach, Germany). Immunoblotting was conducted with primary and secondary antibodies conjugated with horseradish peroxidase and subsequently visualized with ECL enhanced chemoluminescence. Western blots were calibrated with protein standards. To assess sensitivity of the assay, titration experiments were conducted. We determined that cell lysates of 30 000 cells were sufficient to detect MHCII subunits or Ii. In our experiment, we used lysates of 1 million cells for IP and 100 000 cells as lysate controls. The exposure time of the western blots was usually 1 to 5 min. For endoglycosidase treatment, cell lysates were digested with EndoH (200 U) and with PNGase F (100 U) overnight at 37uC as recommended by the supplier (NEB Biolabs, Frankfurt, Germany). For confocal immunofluorescence micros- copy, cells were seeded on coverslips, fixed with 4% PFA, and HLA-Class II-Invariant Chain Complexes permeabilized with 1% Triton X-100. Cells were labeled with CD63 Alexa 488 and with V5 monoclonal antibodies followed by Alexa-594 secondary antibody. Staining was monitored by confocal-fluorescence microscopy (LSM-510 Meta, Zeiss). The size of the optical section was 1 mm. Molecular Modeling A molecular model of a pentameric MHCII-Ii oligomer consisting of a trimeric MHCII-Ii and one bound MHCII heterodimer was generated by combining known experimental structures of a CLIP peptide in complex with HLA-DR3 (pdb1A6A) [6], the known C-terminal trimerisation domain of Ii (residues 118 to 216, pdb1IIE, [3]) and a homology model for the N-terminal coiled-coil structure (residues 7-81) using the SPDBViewer [50] and the Amber molecular modelling software [51]. The N-terminal trimeric coiled-coil structure was built using a known structure (pdb3MO6) as template guided by the leucine repeats in the Ii N-terminal sequence. The connecting loop segments between Ii segments were generated with the SPDBViewer [50]. The subunits were placed manually avoiding steric overlap and allowing insertion of missing loop segments between the C-terminal trimerisation domain, the CLIP sequence and the N-terminal coiled-coil structure employing the loop builder option in SPDBViewer. It is important to emphasize that the structural model represents only one sterically possible structure among many possible alternative loop conformations and variations in the relative subunit arrangement. The complete model was finally energy minimized to remove any sterical overlap employing the Amber package [51]. DNA constructs and antibodies DPa (DPA1*0103), DPb (DPB1*0401) and DQA1*0103 were cloned from dendritic cells generated from blood monocytes. The DP a and b chains were appended with Ab-tags [52]. The DRb (DRB1 0101) chain was tagged with V5-His epitopes (pcDNA3.1 Invitrogen, Karlsruhe, Germany). DRa and DQb contain appended V5 epitopes. TAL-1B5 monoclonal antibody [53] is directed against the cytoplasmic tail of DRa. It was used for immunoprecipitation and for western blotting. Monoclonal antibody I251SB [54] is directed against DRab heterodimers. It does not react with single DR chains. Monoclonal antibodies ISCR3 and 16.23 against DR have been described previously [55– 56]. Serum 35 (S35) was generated by immunizing a rabbit with a DRb fusion protein [57]. Monoclonal antibody LGII-612.14 reacts to DR, DP and DQb chains (purchased from Dr. S. Ferrone, NY). Monoclonal antibody1a3 is specific for DQ and was purchased from MyBioSource (San Diego, CA). Monoclonal antibody Bu43 is directed against the C-terminus, Vic-Y1 to the N-terminus and Bu45 to the trimerisation domain of Ii [58]. V5 and His monoclonal antibodies were purchased from Invitrogen. Sucrose gradient ultracentrifugation The sucrose gradient was poured by gradually mixing 0.4 M with 0.8 M sucrose in 10 mM Tris-buffered saline pH 7.5 containing 0.5% Triton X-100. One ml of Raji cell lysate was placed on top of the gradient and centrifuged at 100.000 g for 50 h. Twenty-four fractions of the gradient were subsequently collected from the top of the tube. The sucrose concentration of the fractions was determined with a refractometer (Fig. 1A. upper panel). By separation of protein standards and subsequent analysis of the fractions by SDS PAGE and Coomassie blue staining, a size calibration of the gradient was achieved (Fig. 1A, lower panel). The protein standard IgG (m.w. 150 kDa), which defines a region with higher molecular weight, was identified in fractions 13 to 16 PLoS ONE | www.plosone.org 11 February 2011 | Volume 6 | Issue 2 | e17257