Intracellular degradation of secretion defect-type mutants of antithrombin is inhibited by proteasomal inhibitors

To examine the cellular basis for secretion defect-type antithrombin deficiency, we expressed two mutants, P --> stop (Pro(429) to stop codon) and Delta Glu (deletion of Glu(313)), Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as Delta Glu mutant was secreted and the total amount of radioactivity was significantly reduced, suggesting an intracellular degradation, The degradation was not inhibited by brefeldin A, indicating it occurring in a preGolgi apparatus, However, the degradation was strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV) and lactacystin, By endoglycosidase H digestion and immunofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER), These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells. (C) 1997 Federation of European Biochemical Societies.