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 (Pro429 to stop codon) and deltaGlu (deletion of Glu313). Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as deltaGlu 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.
Tokunaga
F
F
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Shirotani
H
H
Hara
K
K
Kozuki
D
D
Omura
S
S
Koide
T
T
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000990:Antithrombin III / Q000235:genetics* / Q000378:metabolism*; D020126:Brefeldin A; D002460:Cell Line; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014162:Transfection
D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; C089486:carbobenzoxy-leucyl-leucyl-norvalinal; C067713:lactacystin; D020126:Brefeldin A; D000990:Antithrombin III; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine