PHOSPHATASE INHIBITORS BLOCK IA VIVO BINDING OF PEPTIDES TO CLASS-I MAJOR HISTOCOMPATIBILITY COMPLEX-MOLECULES

Class I major histocompatibility complex (MHC) molecules are heterotri mers of heavy chains, beta(2)-microglobulin, and 8-10 amino acid-long peptides. Assembly of class I MHC molecules into complexes which are s table and can be transported to the cell surface occurs soon after ins ertion of individual subunits into the endoplasmic reticulum (ER). To identify subcellular compartments required for class I MHC assembly, w e studied class I biosynthesis in human cell lines treated with severa l inhibitors of intracellular transport. We found that HLA-B701 molecu les do not assemble in CIR transfectants in which a block in protein t ransport from the ER is established by treatment with phosphatase inhi bitors. In contrast, stable HLA-B701 complexes form in cells in which the ER becomes mixed with the Golgi after treatment with brefeldin A. Neither treatment impaired binding of HLA-B701 to the ER-resident prot ein calnexin, and unassembled heavy chains in phosphatase-inhibited ce lls showed prolonged association with calnexin. In addition, the mouse class I molecule H-2D(b), which binds beta(2)-microglobulin in human T2 cells in the absence of transporter of antigenic peptides, formed c omplexes in CIR cell transfectants treated with phosphatase inhibitors . Taken together, these data demonstrate that phosphatase inhibitors d o not prevent assembly of class I heavy chain-beta(2)-microglobulin di mers, but instead interfere with peptide loading. These results are co nsistent with the possibility that class I MHC molecules are transport ed from their initial site of insertion into the rough ER before bindi ng peptides, or alternatively that peptide loading mediated by transpo rter of antigenic peptides is blocked by phosphatase inhibitors.