Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality control.
The rate-limiting step in protein secretion is folding, which occurs in the endoplasmic reticulum (ER) lumen, and almost all secreted proteins contain disulfide bonds that form in the ER and stabilize the native state. Secreted proteins unable to fold may aggregate or they may be subject to ER-associated protein degradation. To examine the fate of aberrant forms of a well characterized, disulfide-bonded secreted protein, we expressed bovine pancreatic trypsin inhibitor in yeast. Bovine pancreatic trypsin inhibitor is a single domain, 58-amino acid polypeptide containing three disulfide bonds, and yeast cells secrete the wild type protein. In contrast, the Y35L mutant, which folds rapidly but is unstable, remains soluble and is not secreted. Surprisingly, the proteolysis of Y35L is unaffected in yeast containing mutations in genes encoding factors required for ER-associated protein degradation and is stable if artificially retained in the ER. Rather, Y35L is diverted from the Golgi to the vacuole and degraded. Because only the mutant protein is quantitatively proteolyzed these data suggest that a post-ER quality control check-point diverts unstable proteins to the vacuole for degradation.
Coughlan
Christina M
CM
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Walker
Jennifer L
JL
Cochran
Jared C
JC
Wittrup
K Dane
KD
Brodsky
Jeffrey L
JL
D000818:Animals; D007611:Aprotinin / Q000235:genetics* / Q000378:metabolism*; D037281:Calnexin / Q000737:chemistry; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000378:metabolism; D005838:Genotype; D006056:Golgi Apparatus / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism*
D004220:Disulfides; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D011134:Polysaccharides; D011500:Protein Synthesis Inhibitors; D003208:Concanavalin A; D037281:Calnexin; D013395:Sucrose; D007611:Aprotinin; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex