APOLIPOPROTEIN-B IS INTRACELLULARLY ASSOCIATED WITH AN ER-60 PROTEASEHOMOLOG IN HEPG2 CELLS

Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic re ticulum (ER)-localized proteases (ER-60 and ER-72) were recently purif ied from rat liver, We used an antibody to rat ER-60 to investigate th e possible role of this protease in apolipoprotein B (apoB) degradatio n, First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58-60 kDa. The ER-60 h omologue was mostly associated with the luminal contents of HepG2 micr osomes, Evidence from co-immunoprecipitation and cross-linking experim ents appear to suggest that the ER-60 homologue in HepG2 cells is asso ciated with apoB intracellularly. A small pool of apoB was recovered w hen HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody, Furthermore, cross-linking of permeabilized cells with dith iobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells, Three polypeptides with mole cular masses of 78, 66, and 50 kDa were consistently found to be assoc iated with apoB as well as the 58-kDa ER-60 homologue, The 78-kDa prot ein associated with both apoB and ER-60 appeared to represent immunogl obulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody, Cross-linking and immunoblotting experimen ts suggested the association of the 78-kDa BiP with both the 58-kDa ER -60 homologue as well as the 550-kDa apoB. In summary, the data sugges ts that HepG2 cells contain a 58-kDa protein which is homologous to th e rat liver ER-60 in size, antigenecity, and intracellular localizatio n, The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chapero nes such as BiP, We hypothesize that the ER-60 homologue may be involv ed in the degradation of apoB in the ER lumen of HepG2 cells.