LYSOSOMAL DEGRADATION AND SORTING OF APOLIPOPROTEIN-E IN MACROPHAGES

We previously reported that a substantial amount of newly synthesized apoE in mouse macrophages is degraded prior to secretion; a portion of this pool of apoE can be rescued by the addition of HDL(3) to the inc ubation medium. In the present studies, the location and nature of the intracellular degradation of apoE were more closely examined. Inhibit ors of protein trafficking (brefeldin A) as well as a number of protea se inhibitors were used. The experiments using brefeldin A (5 mu g/ml) clearly established that neither the endoplasmic reticulum nor the Go lgi complex are the sites of apoE degradation. Using a pulse-chase des ign, [S-35]apoE cannot be chased out in the presence of brefeldin A an d remains undegraded within the cell. The accumulated apoE lacks the s ialic acid residues, indicating that this final stage of processing mu st occur in the trans-Golgi network or later. Lysosomotropic agents, a mmonium chloride and chloroquine, on the other hand, inhibit apoE degr adation by over 70 and 80%, respectively, while total cell protein deg radation remains unaffected. Similarly, a cocktail consisting of four lysosomal protease inhibitors (pepstatin, E-64, chymostatin, and antip ain), inhibits specifically apoE degradation by over 60%. In contrast, ALLN, an inhibitor of Ca2+-dependent cysteine proteases, has a modera te effect on apoE degradation (30% inhibition) and a more pronounced e ffect on total protein degradation. These data suggest that the site o f intracellular apoE degradation in the macrophage is the lysosome. Th ese conclusions are supported by light and electron microscopy of macr ophages, clearly showing the presence of immunoreactive apoE (along wi th cathepsin D) in the endosomal /lysosomal compartment of control and lysosomotropic agent-treated cells. In contrast, little or no labelin g is seen in this compartment in brefeldin A-treated cells. At lower c oncentrations of the lysosomotropic agents, the extent of inhibition o f apoE degradation is compensated for by its increased secretion, in a manner analogous to the effect of these agents on lysosomal enzymes. Higher concentrations of these agents, which lead to a profound inhibi tion of apoE degradation, also specifically block apoE secretion. The block in apoE secretion in the presence of high concentrations of chlo roquine leads to undiminished or higher concentrations of immunoreacti ve apoE in the endosomal/lysosomal compartment, suggesting that apoE i s targeted for lysosomal degradation directly, without prior secretion or surface association. These data strongly suggest pH-dependent sort ing of apoE in macrophages to the degradative and secretory pathways a nd imply a protein-protein interaction in the process.