CALNEXIN AND OTHER FACTORS THAT ALTER TRANSLOCATION AFFECT THE RAPID BINDING OF UBIQUITIN TO APO-B IN THE SEC61 COMPLEX

Several secretory proteins, including apolipoprotein B, have been show n to undergo degradation by proteasomes. We found that the rapid degra dation of nascent apolipoprotein B in HepG2 cells was diminished but n ot abolished by the addition of any of three different inhibitors of p roteasomes, Ubiquitin is conjugated to apolipoprotein B that is not as sembled with sufficient lipids either during or soon after synthesis, In addition, we found that apolipoprotein B that has entered the endop lasmic reticulum sufficiently to become glycosylated can be degraded b y proteasomes. Furthermore, we detected ubiquitin-apolipoprotein B tha t is associated with the Sec61 complex, the major constituent of the t ranslocational channel. Treatment of cells with monomethylethanolamine or dithiothreitol decreased the translocation of apolipoprotein B and increased the proportion of ubiquitin-conjugated molecules associated with Sec61. Conversely, treatment of cells with oleic acid, which inc reased the proportion of translocated apolipoprotein B, decreased the amount of ubiquitin-apolipoprotein B in the Sec61 complex. Finally, we found that inhibition of the interaction between calnexin and apolipo protein B decreases the translocation of apolipoprotein B, increases t he ubiquitin-apolipoprotein B in the Sec61 complex, and increases the proteasomal degradation of glycosylated apolipoprotein B, Thus, ubiqui tin can be attached to unassembled apolipoprotein B in the Sec61 compl ex, and this process is affected by factors including calnexin that al ter the translocation of apolipoprotein B.