STRUCTURAL-ANALYSIS OF PROTEOLYTIC PRODUCTS OF MHC CLASS-II-INVARIANTCHAIN COMPLEXES GENERATED IN-VIVO

The MHC class II alphabeta heterodimer associates with invariant (I) c hain in the endoplasmic reticulum and remains associated until the com plex reaches a post-Golgi compartment. During early stages of transpor t, I chain blocks peptide binding to alphabeta dimers. I chain is prot eolytically cleaved in a post-Golgi compartment releasing alphabeta di mers that can bind antigenic peptides and transport them to the cell s urface. Human B lymphoblastoid cell lines grown in leupeptin, a sulfhy dryl protease inhibitor, accumulate a partial proteolytic product of t he I chain called leupeptin-induced protein (LIP). LIP remains associa ted with alphabeta dimers. We find, using chemical cross-linking, sucr ose gradient sedimentation, and size exclusion chromatography, that th e alphabetaLIP complex retains the nine-subunit structure described fo r alphabetaI complexes. Unlike the alphabetaI complex, in certain dete rgents the alphabetaLIP nonamer is unstable and dissociates into trime rs containing one alpha, beta, and LIP molecule. This finding emphasiz es the reported stoichiometry of the alphabetaI complex as a nine-subu nit structure comprised of three alphabetaI trimers. Also, these data indicate that the region(s) of I chain necessary for retaining the non americ structure lie within the LIP fragment, but that domains to the C-terminus of the LIP cleavage site act to further stabilize the nine chain structure. In addition, alphabetaI complexes containing forms of human I chain encoding the p35/p43 N-terminal cytoplasmic extension r esponsible for endoplasmic reticulum retention can transport to post-G olgi proteolytic compartments where LIP is formed.