engleski

Nonconformed and misfolded MHC-I molecules are sorted into lipid rafts

Problem: The aim of our study was to determine the localization of conformed and non-conformed MHC class I molecules (Kd, Dd and Ld) in different membrane microdomains of non-polarized murine cell lines (P815 mastocytoma cell line, and Balb 3T3 and L-Ld fibroblast cell line). Material and methods: Transformation of Ld molecules into the empty conformation (misfolded molecules) was achieved by short acidification of cell culture medium. The localization of cellsurface and internalized MHC-I molecules was followed by confocal microscopy after surface labeling with monoclonal antibodies (mAbs). Endocytosis of Ld molecules was also determined by flow cytometry. Cholesterol depletion by filipin and nystatin was used to examine the role of cholesterol dependent endocytic pathways, and MHC-I molecules were determined by flow cytometry, confocal microscopy, and cell surface biotinylation followed by subsequent immunoprecipitation and SDS-PAGE. Finally, localization of surface molecules in detergent insoluble membrane domains was determined by short ice-cold treatment with nonionic detergents (Triton X-100, CHAPS, Brij 96 and Tween 20) and confocal microscopy, or cell surface biotinylation followed by subsequent immunoprecipitation and SDS-PAGE. Results: We have found that conformed (Kd, Dd and Ld) and non-conformed, either normally expressed Ld molecules at the cell surface or misfolded (Dd and Ld) molecules predominantly localize at different sites of the plasma membrane. Endocytosis of non-conformed molecules, but not conformed, , is cholesterol dependent because it was inhibited by cholesterol-sequestering agents (filipin and nystatin). Howerver, non-conformed molecules did not colocalize with caveolin-1, a marker of caveolae and caveolar endocytosis, although they partially colocalized with cholera toxin B subunit (CTB). Resident intracellular nonconformed Ld molecules also partially collocalized with GM1 (binding of CTB). Nonconformed Ld molecules were resistant to extraction by nonionic detergents (primary with Triton X-100), both at the cell surface and at the intracellular vesicles after endocytosis. This is similar to GM1, a marker of lipid rafts, but different to conformed MHC-I molecules and transferrin receptor. Conclusion: Nonconformed MHC-I molecules localize in detergent resistant domains (lipid rafts) and are internalized by cholesterol dependent pathway. In contrast, conformed Ld molecules do not localize in the lipid rafts and are internalized via bulk pathway.

lipid rafts; endocytosis; MHC-I molecules; conformed and non-conformed molecules

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