HLA-DM AND MHC CLASS-II MOLECULES CO-DISTRIBUTE WITH PEPTIDASE-CONTAINING LYSOSOMAL SUBCOMPARTMENTS

MHC class II molecules associate with peptides in the endocytic pathwa y, Different endosomal locations for peptide loading of class II molec ules, varying from early endosomes (EE) to lysosomes, have been assign ed on the basis of subcellular fractionation experiments, We have dete rmined the intracellular location of HLA-DM, a molecule that supports peptide loading of class II molecules, by separating vesicles from the melanoma cell line Mel JuSo on the basis of buoying density and surfa ce charge, In both fractionations, HLA-DM co-fractionated with a lysos omal compartment containing beta-hexosaminidase (beta-hex) activity an d not with endosomes, Further analysis showed that HLA-DM mainly co-fr actionated with a sub-lysosomal structure characterized by a relative low density and containing both pro- and mature cathepsin D and MHC cl ass II molecules, Fluid phase markers first enter this compartment bef ore entering high-density lysosomes that contain exclusively mature ca thepsin D, some HLA-DM and no detectable MHC class II molecules. Final ly we determined the intracellular location of neutral and acidic pept idases. Whereas neutral peptidase activity was detected in the endopla smic reticulum and/or plasma membrane fractions, acidic peptidase acti vity exclusively migrated at the position of HLA-DM containing lysosom al vesicles, Our results show that class II molecules co-migrate with HLA-DM, pro- and mature cathepsin D, beta-hex and acidic peptidase act ivity, HLA-DM, cathepsin D and class II molecules were not observed at the position of EE. Our data suggest that HLA-DM-mediated peptide loa ding of class II molecules occurs in a lysosomal subcompartment.