Differential Degradation of the Three Fibrinogen Chains by Proteasomes: Involvement of Sec61p and Cytosolic Hsp70

HepG2 cells, which synthesize and secrete fibrinogen, accumulate surplus Aα and γ chains. The nonsecreted fibrinogen chains are degraded both by proteasomes and lysosomes, with unassembled chains primarily degraded by proteasomes and an Aα–γ complex by lysosomes. To further determine the mechanisms by which unassembled fibrinogen chains are degraded, and to explain the pools of Aα and γ chains that occur in HepG2 cells, the association of fibrinogen chains with Sec61β, a component of the translocon, and with a cytosol chaperone, Hsp70, was studied in both HepG2 cells and COS cells expressing single fibrinogen chains. Retrotranslocation from the lumen of the endoplasmic reticulum was shown by treatment with MG132, a proteasome inhibitor. MG132 caused glycosylated Bβ to accumulate on Sec61β in COS cells expressing Bβ and acted similarly with all three fibrinogen chains in HepG2 cells. In HepG2 cells, Bβ was associated with Sec61β ahead of Aα and γ chains, suggesting that pools of Aα and γ chains may be caused by unequal rates of retrotranslocation. In COS cells, retrotranslocation into the cytoplasm was demonstrated by the ATP-sensitive association of ubiquitinylated Aα, Bβ, and γ chains bound to Hsp70. More Aα and γ than Bβ accumulated on Hsp70 of HepG2 cells, consistent with more rapid degradation of Bβ. Overexpression of Hsp70 in HepG2 cells resulted in decreased secretion, but not synthesis, of fibrinogen. Decreased secretion may be due to enhanced degradation of unassembled fibrinogen chains, indicating that proteolysis by proteasomes might regulate both the intracellular pools of fibrinogen chains and fibrinogen secretion.