ASSEMBLY OF MHC CLASS-I MOLECULES WITH BIOSYNTHESIZED ENDOPLASMIC RETICULUM-TARGETED PEPTIDES IS INEFFICIENT IN INSECT CELLS AND CAN BE ENHANCED BY PROTEASE INHIBITORS

To study the requirements for assembly of MHC class I molecules with a ntigenic peptides in the endoplasmic reticulum (ER), we studied Ag pro cessing in insect cells. Insects lack a class I recognition system, an d their cells therefore provide a ''blank slate'' for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2K(b) heavy chain, mouse beta(2)-microglobulin , and an ER-targeted version of a peptide corresponding to Ova(257-264 ) were expressed in insect cells using recombinant vaccinia viruses, C ell surface expression of K-b-OVA(257-264) complexes was quantitated u sing a recently described complex-specific mAb (25-D1.16), Relative to TAP deficient human cells, insect cells expressed comparable levels o f native, peptide-receptive cell surface K-b molecules, but generated cell surface K-b-OVA(257-264) complexes at least 20-fold less efficien tly from ER-targeted peptides. The inefficient assembly of K-b-OVA(257 -264) complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking t apasin expressed endogenously synthesized K-b-OVA(257-264) complexes a t levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not det ectably enhance the expression of K-b-OVA(257-264) complexes, The asse mbly of K-b-OVA(257-264) complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor, These fin dings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cel ls and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degrada tion.