Nascent Lipidated Apolipoprotein B Is Transported to the Golgi as an Incompletely Folded Intermediate as Probed by Its Association with Network of Endoplasmic Reticulum Molecular Chaperones, GRP94, ERp72, BiP, Calreticulin, and Cyclophilin B*

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate is dependent on the assistance of molecular chaperones for its subsequent folding steps. To that end, microsomes were prepared from HepG2 cells and luminal contents were subjected to KBr density gradient centrifugation. Immunoprecipitation of apoB followed by Western blotting showed that the luminal pool floated at a density of 1.12 g/ml and, like the membrane-bound pool, was associated with GRP94, ERp72, BiP, calreticulin, and cyclophilin B. Except for calreticulin, chaperone/apoB ratio in the lumen was severalfold higher than that in the membrane, suggesting a role for these chaperones both in facilitating the release of the primordial intermediate into the ER lumen and in providing stability. Subcellular fractionation on sucrose gradients showed that apoB in the Golgi was associated with the same array of chaperones as the pool of apoB recovered from heavy microsomes containing the ER, except that chaperone/apoB ratio was lower. KBr density gradient fractionation showed that the major pool of luminal apoB in the Golgi was recovered from 1.02 < d < 1.08 g/ml, whereas apoB in ER was recovered primarily from 1.08 < d < 1.2 g/ml. Both fractions were associated with the same spectrum of chaperones. Together with the finding that GRP94 was found associated with sialylated apoB, we conclude that correct folding of apoB is dependent on the assistance of molecular chaperone, which play multiple roles in its maturation throughout the secretory pathway including distal compartments such as the trans-Golgi network.