A cytosolic molecular chaperone, Hsp70, plays a role during the ER-associated translocation and degradation of a secretory protein, apoprotein B100

This thesis addresses the mechanisms for the regulation of apoprotein B100 (apoB)-containing lipoprotein secretion from cultured hepatic cells. The first study demonstrated that nascent apoB was bound to a cytosolic chaperone protein, heat shock protein 72/73 (Hsp70). This apoB-hsp70 binding was transient, adenosine triphosphate-sensitive, and was correlated with the extent of translocation of apoB across the endoplasmic reticulum (ER) membrane. The N-terminal 16% of apoB (apoB 16), which displays efficient translocation in stably transfected CHO cells, showed minimal binding to Hsp70. The N-terminal 50% of apoB (apoB50), which is poorly translocated in CHO cells, was found to bind significantly to Hsp70. These results suggested that nascent apoB is transiently exposed to the cytosol, and that Hsp70 may function as a molecular chaperone for apoB during the ER translocation process.;The second study demonstrated that the protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), markedly and selectively increased Hsp70. The modulation of Hsp70 synthesis by ALLN resulted from a rapid and marked increase in the transcription of the hsp72 gene due to induced trimerization of the heat shock transcriptional factor (HSF1). These effects of ALLN appeared to require de novo protein synthesis, and were reproduced with other proteasome-specific inhibitors, suggesting that a rapidly turning-over protein that is normally degraded by proteasomes may be involved in the regulation of Hsp70 synthesis via effects on HSF1.;To explore the role that Hsp70 plays in apoB translocation and degradation, the third study demonstrated that increasing the expression of Hsp70 in HepG2 cells, either by Herbimycin A treatment or by transient transfection with rat hsp72 cDNA, promoted apoB degradation. ApoB degradation, both normally and after Hsp70 induction, was blocked by inhibitors of the proteasome. In addition, apoB was shown to be conjugated with ubiquitin. The apoB that accumulated after proteasome inhibition was ER-associated and could be assembled into lipoproteins and secreted if newly lipid synthesis was stimulated. These results indicated that apoB is degraded by a proteolytic pathway involving Hsp70, ubiquitin, and the proteasome.;Additional studies demonstrated that increased levels of Hsp70 were associated with increased apoB ubiquitination (Ubn). Inhibition of microsomal triglyceride transfer protein, an ER-luminal chaperone, was associated with both complete blockade of apoB secretion and increased apoB Ubn. ApoB50 expressed in CHO cells displayed heavy Ubn without secretion, whereas apoB 16 had little or no Ubn with significant secretion. Finally, with use of puromycin pretreatment to synchronize apoB translation, the immature apoB polypeptides that had not reached the full length clearly demonstrated Ubn. These results indicate that, in HepG2 cells, apoB Ubn can occur co-translationally and is regulated by the efficiency of ER-translocation.