engleski

Nonconformed Ld molecules are sorted into lipid rafts for endocytosis

Problem: The aim of our study was to determine the presence of conformed (full) and non- conformed (empty) Ld molecules in lipid rafts at the plasma membrane of non-polarized cell lines (P815 mastocytoma cell line and Balb 3T3 fibroblast cell line). Material and methods: Endocytosis of Ld molecules was followed in a model of spontaneous internalization by using cycloheximide (an inhibitor of protein synthesis) or induced by binding of monoclonal antibodies. Transformation of Ld molecules into the empty conformation was achieved by short acidification of cell culture medium. The cell surface expression of Ld molecules was determined by flow cytometry, and intracellular colocalization with markers of the endocytic pathway by confocal microscopy. Cholesterol depletion by filipin and nystatin was used to examine the role of cholesterol dependent endocytic pathways, and Ld molecules were determined by flow cytometry, confocal microscopy, and cell surface biotinylation followed by subsequent immunoprecipitation and SDS-PAGE. Finally, localization of Ld molecules in detergent insoluble membrane domains was determined by short ice-cold Triton X treatment and confocal microscopy. Results: We found that both full and empty Ld molecules are internalized and subsequently degraded on both cell lines with similar kinetics in two different models of endocytosis. The endocytosis of empty, but not of full Ld molecules is cholesterol dependent because it was inhibited by treatment with cholesterol-sequestering agents (filipin and nystatin). However, empty Ld molecules did not colocalize with caveolin – a marker of caveolae and caveolar endocytosis, although they partially colocalized with cholera toxin B subunit (CTB). After acid treatment cell surface full Ld molecules were transformed into empty conformation. They were resistant to Triton X extraction, which is similar to GM1– a marker of lipid rafts, but different to full Ld molecules and transferrin receptor. Conclusion: Nonconformed Ld molecules localize in detergent resistant domains (DRG) - 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; MHC class I molecules; endocytosis; protein sorting

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