Apoprotein B degradation is promoted by the molecular chaperones hsp90 andhsp70

Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipop roteins, The net production of apoB can be regulated by presecretory degrad ation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a c ell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated , and ubiquitinylated, Subsequent incubation with rat hepatic cytosol led t o proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay . GA increased the recovery of microsomal apoB48 similar to3-fold and disru pted the interaction between hsp90 and apoB48, Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradatio n was reconstituted in vitro using cytosol prepared from wild type yeast. N otably, degradation was attenuated when apoB48-containing microsomes were i ncubated with cytosol supplemented with GA or with cytosol prepared from ye ast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between en doplasmic reticulum-associated apoB and components of the proteasomal pathw ay, perhaps in cooperation with hsp70.