HLA-DM IS PRESENT IN ONE-5TH THE AMOUNT OF HLA-DR IN THE CLASS-II PEPTIDE-LOADING COMPARTMENT WHERE IT ASSOCIATES WITH LEUPEPTIN-INDUCED PEPTIDE (LIP)-HLA-DR COMPLEXES

HLA-DM has been shown in vitro to catalyze the release of invariant ch ain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Pre vious studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociat es from class II molecules and antigenic peptides are bound. Here we c haracterize the expression of DM in vivo in subcellular fractions cont aining the IIPLC. Using quantitative immunoblotting, we show that in t he cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is pr esent in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1DM. This molar ratio of DM to DR i n the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and ge netic evidence that DM associates with complexes which contain II frag ments and class II molecules in the IIPLC. Such complexes are only obs erved in leupeptin-treated cells in which II fails to be completely de graded and complexes containing the leupeptin-induced fragment of ii ( LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extreme ly transient under normal conditions.