Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

• Published in: Human immunology Vol. 53; no. 2; pp. 129 - 139
• Main Authors: Bresnahan, Patricia A., Barber, Linda D., Brodsky, Frances M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1997
• Subjects: ATP-Binding Cassette Sub-Family B Member 2; ATP-Binding Cassette Transporters - metabolism; Biological Transport; Cell Compartmentation - immunology; Centrifugation, Density Gradient; Endoplasmic Reticulum, Rough - metabolism; Histocompatibility Antigens Class I - metabolism; Humans; Lymphoma, B-Cell; Subcellular Fractions - immunology; Subcellular Fractions - metabolism; Tumor Cells, Cultured; PBS; TAP; SRβ; FBS; MoAb; MHC; IC; RER
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/S0198-8859(97)00001-3

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β 2-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.