engleski

Endocytic pathway of MHC class I molecules is conformation dependent

MHC class I molecules are expressed in a cell in two conformations: either as full, composed of properly folded heavy chain,  2 – microglobulin ( 2m) and peptide, or as empty, represented either by non-conformed heavy chains or heterodimer of weakly associated non-conformed heavy chain and  2m. The aim of our study was to compare the internalization mechanisms and endocytic trafficking of conformed and non-conformed murine MHC class I (MHC-I) molecules (Kd, Dd and Ld) on non-polarized cell lines P815 mastocytoma cell line and Balb 3T3 fibroblast cell lines. Endocytosis and intracellular trafficking of MHC-I molecules were followed in the model of spontaneous internalization after cycloheximide treatment, or after surface binding of monoclonal antibodies (mAbs). Endocytic pathway was dissected by treatment with inhibitors of endocytosis and endosomal acidification. Transformation of MHC class I molecules into misfolded conformation was achieved by short acidification of cell culture medium. Cell surface expression of MHC-I molecules was determined by flow cytometry and intracellular expression by confocal microscopy. Cell surface biotinylation, followed by immunoprecipitation and SDS-PAGE, was used to determine the kinetics of degradation. Non-conformed Dd and Ld molecules are localized in the lipid rafts, and are internalized by cholesterol dependent mechanism that is not caveolar. In contrast, conformed class I molecules are internalized by the bulk pathway and are not localized in the lipid rafts. After internalization, both conformed and non-conformed molecules were found in EEA1+ compartments, but not before 10-20 minutes after internalization. However, conformed MHC molecules are more intensively recycled and colocalized with Rab11 (the marker of early endosomes), in difference to nonconformed MHC-I molecules that are directed to an endosomal retention compartment (empty Ld compartment - ELC), a place where empty Ld molecules normally accumulate. Finally, both types of class I molecules are degraded in lysosomes, but with different kinetics. According to our results, conformed and non-conformed MHC-I molecules are internalized by different mechanisms, but their endocytic pathways are intersected in sorting endosomes and lysosomes. Conformed MHC-I molecules are more intensively recycled, while non-conformed molecules are retained in separate compartment. Finally, both conformation are directed into lysosomes for degradation.

endocytosis; MHC-I molecules; vesicular transport

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