LOCALIZATION OF CLASS-I HISTOCOMPATIBILITY MOLECULE ASSEMBLY BY SUBFRACTIONATION OF THE EARLY SECRETORY PATHWAY

Class I molecules of the major histocompatibility complex bind peptide s derived from cytosolic proteins and display them on the cell surface . This function alerts cytotoxic T cells to the presence of intracellu lar pathogens. Class I molecule assembly requires the association of t he heavy chain with beta(2)-microglobulin, accompanied by peptide load ing via specific transporters. This study localizes where these assemb ly steps rake place, using monoclonal antibodies recognizing class I m olecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded cl ass I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molec ules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RE R) membranes. These data suggest that subunit assembly and peptide acq uisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, w hich contained comparatively less peptide-loaded molecules than the hi gh density RER fractions, at steady state. Following a 15 degrees C te mperature block, class I molecules accumulated in these less dense mem brane fractions, indicating that these fractions represent the interme diate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules r ecycling to the RER was detected, suggesting empty molecules recycle t o acquire peptide. Human Immunology 53, 129-139 (1937). (C) American S ociety for Histocompatibility and Immunogenetics, 1997.