^^^^^^S^B^S^^^S^P^^^S^S^S^S^S! 223 (1996); Y. Maru, D. E. Afar, O. N. Witte, M. Shibuya, J. Biol. Chem. 271, 15353 (1996). 18. L. S. Friedman etal.,Am. J. Hum. Genet. 57, 1284 (1995); O. Johannsson etal.j'bid. 58, 441 (1996). 19. B. M. Benton etal.,Mol. Cell. Biol. 10, 353 (1990). 20. R. D. Gietz and A. Sugino, Gene 74, 527 (1988). 21. Yeast strains used were BYB69 (MA7a ade2-1 can1-100 his3-11,15 leu2-3,112 lys2A::hisG trp1-1 ura3-1 ste5A::LYS2), BYB84 {MATagal2 Ieu2 prbl- 1122pep4-3prc1-407trp1 ura3-52 ste5A), BYB88 (MA 7a ade2-101 oc his3-A200 leu2A-1 Iys2-801 am trp1-A63 ura3-52 ste4A::TRP1 ste5A::LYS2), and DC17(MATa/7/s7). 22. The CEN plasmid (pCJ117) expressing (His)6- and Myc epitope-tagged STE5 + from the GAL1 pro- moter is described in detail elsewhere {15). A mul- ticopy plasmid (pCJ6) expressing the same con- struct was produced by inserting the 3.3-kb Bam Hl-Bam HI S7E5-containing fragment from pCJ117 into the Bam HI site in the vector, YEp352Gal {19). pCJ93 expresses the same con- struct from the authentic STE5 promoter in the vector, YCplac33 {20), and was engineered with polymerase chain reaction (PCR) to have the se- quence, 5'-CATATGATG-3\ immediately up- stream and in-frame with the first codon of the (His)6 tag. The double mutant allele, ste5(C177A C180A), used in most of the experiments present- ed here was produced by PCR amplification using a primer encoding the sequence 5'-AAC- GCGTCTGC-TACGTTAGCT-3', in which the indi- cated bases (underlined) were altered to convert the Cys codons at positions 177 and 180 to Ala codons (and in which the silent mutations, indicat- ed by boldface, were introduced to create an Mlu I site). The mutated segment was then used to re- place the corresponding fragment in the other S7E5-containing plasmids to generate constructs in which Ste5(C177A C180A) was expressed from the STE5 promoter on a CEN plasmid (pCJ70), from the GAL1 promoter on a CEN plasmid (pCJ119), and from the GAL1 promoter on a mul- ticopy plasmid (pCJ48). 23. F. Sherman, G. R. Fink, J. B. Hicks, Methods in Yeast Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). 24. Protease-deficient strain BYB84 {21), carrying vec- tor alone or expressing from the GAL1 promoter NH2-terminally c-Myc-tagged versions of either normal Ste5 or the RING-H2 domain mutant {22), and also carrying either a CEN vector alone, or the same vector expressing STE11, STE7, or STE4 from the GAL1 promoter, or expressing FUS3 from the endogenous FUS3 promoter on a 2 jxm DNA- based vector, were grown under selection in SCRaf medium {15) to A6 : 0.6. The cultures were induced by the addition of Gal to a final concentra- tion of 2% and incubated for an additional 2 to 3 hours. The cells were collected, lysed, clarified, and the resulting extracts were subjected to immuno- precipitation {15). The resulting immune complexes were resuspended in 1 x SDS-PAGE sample buff- er, boiled for 5 min, and then resolved by SDS- PAGE. After transfer onto Immobilon-P mem- branes (Millipore) using semidry transfer apparatus (Bio-Rad), proteins were detected by immunoblot- ting with rabbit polyclonal antisera to Ste11, Ste7, Fus3, Ste4, and Ste5, as appropriate. 25. Qualitative mating tests were performed by patch- ing the MATa strains to be tested on appropriate selective medium, and then replica-plating onto a lawn of DC17 on YP medium containing either 2% Gal/0.2% Sue or 2% Glc (depending on the pro- moter used for STE5 expression) at 30°C over- night. The resulting mating plates then were repli- ca-plated onto a minimal medium [synthetic com- plete (SC)] {23) selective for diploids and further incubated at 30°C overnight. 26. The GST fusions were generated as follows. A 640- bp fragment encoding S. japonicum GST was generated by PCR with a pGEX vector (Pharmacia) as the template and appropriate primers to install Ase I sites at both the 5' and 3' ends of the prod- uct, and inserted into'the Nde I site in-frame and downstream of codon 913 of the STE5 coding se- quence in pCJ117 {22), creating a junction (5'- CATAATATGTCC-3') encoding His gi2Asngi3 Met- Ser (where Met is the first residue of GST), thus yielding pCJ148. pCJ149, expressing the Ste5(C177A C180A)-GST fusion, was created from pCJ119 in an analogous fashion. The translation stop codon in both pCJ148 and pCJ149 is provid- ed by the natural TAG at the end of the STE5 coding sequence. 27. MATa strains to be tested were grown in an appro- priate selective medium (SC) {23) containing either 2% glucose (Glc) or 2% raffinose (Raf), depending on the promoter regulating STE5 expression. Strains carrying plasmids expressing STE5 con- structs from the GAL1 promoter were induced by addition of galactose (Gal) to a final concentration of 2% and incubation for 60 min before dilution and were plated on a medium containing 2% Gal and 0.2% sucrose (Sue). Samples (0.2 ml) of serial di- lutions of the MATa strains were mixed, in triplicate, with 0.6 ml of a culture of a MA7a tester strain (DC17) that had been grown to midexponential phase in yeast extract-peptone (YP) {23) medium. Portions (0.4 ml) of these mixtures were plated on a medium lacking the appropriate supplements to select for diploids and incubated at 30°C for 36 to 40 hours. Corresponding dilutions of the MATa strains were also plated to determine the total num- ber of viable haploids. Mating efficiencies were cal- culated as the ratio of diploid cells formed to the total number of input MATa haploids. 28. We thank J. Schultz, M. S. Hasson, R. L Freedman, and E. T. Barfod for early contributions, L. Bardwell and J. G. Cook for critical reading of the manuscript, and T. Durfee for intellectual contributions, helpful discussions, and enthusiastic interest. Supported by grant GM21841 from NIH (J.T.), postdoctoral fellow- ship PF-3785 from the American Cancer Society (C.I.), by National Cancer Institute postdoctoral train- eeship CA09041 and NRSA postdoctoral fellowship from NIH (N.D.), and by resources provided by the Berkeley campus Cancer Research Laboratory. 7 July 1997; 21 August 1997 Negative Regulation by HLA-DO of MHC Class ll-Restricted Antigen Processing Lisa K. Denzin, Derek B. Sant'Angelo, Craig Hammond, Michael J. Surman, Peter Cresswell* HLA-DM is a major histocompatibility complex (MHC) class ll-like molecule that facil- itates antigen processing by catalyzing the exchange of invariant chain-derived peptides (CLIP) from class II molecules for antigenic peptides. HLA-DO is a second class ll-like molecule that physically associates with HLA-DM in B cells. HLA-DO was shown to block HLA-DM function. Purified HLA-DM-DO complexes could not promote peptide ex- change in vitro. Expression of HLA-DO in a class l l + and DM + , DO - human T cell line caused the accumulation of class II—CLIP complexes, indicating that HLA-DO blocked DM function in vivo and suggesting that HLA-DO is an important modulator of class II—restricted antigen processing. MHC class II molecules assemble in the endoplasmic reticulum (ER) as a nonameric complex consisting of an invariant chain trimer associated with three class II a(3 dimers (J). Signals in the invariant chain cytoplasmic domain direct the complex into the endocytic pathway, where invariant chain degradation results in the transient formation of a class II a (3 dimer with a residual fragment of the invariant chain, CLIP (class Il-associated invariant chain peptides), in the peptide-binding groove (2). The interaction of the a(3-CLIP com- plex in the antigen-processing compart- ment, or MHC class II compartment (MIIC) (3), with a second class II—like molecule, called HLA-DM in humans and H-2M in mice, induces CLIP dissociation (4, 5). Association of empty a(3 dimers with DM stabilizes them until high-affin- ity peptides derived from internalized pro- teins can bind (6, 7). Mature a(3-peptide Section of Immunobiology, Howard Hughes Medical In- stitute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA. *To whom correspondence should be addressed. E-mail: peter.cresswell@qm.yale.edu complexes then leave the endocytic path- way and are expressed on the cell surface. Another class II—like molecule, HLA- DO, expressed only in B cells and thymic epithelium (8-J2), physically associates with DM in the ER and during and after transport to the MIIC (8). We therefore investigated what the effect of DO associ- ation might be on the ability of DM to catalyze CLIP dissociation and peptide loading. HLA-DM was affinity-purified (4) from the Burkitt's lymphoma B cell line Raji and from the DO-negative DM- transfectant T2/DM (13). Protein immu- noblotting with rabbit antisera specific for the DM and DO-(3 chain cytoplasmic do- mains (14) showed that the purified ma- terial from Raji contained DO, whereas that from T2/DM did not (Fig. 1A). DM- DO association was maintained through extensive washing of the monoclonal an- tibody (mAb) MaP.DMB/c affinity col- umn with sodium deoxycholate. Thus, the interaction is qualitatively different from that of DM with conventional class II molecules, which is retained only in mild detergents such as CHAPS or digitonin 106 SCIENCE • VOL. 278 • 3 OCTOBER 1997 • www.sciencemag.org