title: The proteasome is involved in the degradation of different aquaporin-2 mutants causing nephrogenic diabetes insipidus.
abstract: Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t(1/2) of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t(1/2) = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t(1/2) = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants.
authors.affiliation: Department of Pathology, Division of Cell and Molecular Pathology, University of Zürich, Zürich, Switzerland.
authors.forename: Kiyoko
authors.initials: K
authors.lastname: Hirano
authors.forename: Christian
authors.initials: C
authors.lastname: Zuber
authors.forename: Jürgen
authors.initials: J
authors.lastname: Roth
authors.forename: Martin
authors.initials: M
authors.lastname: Ziak
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D007150:Immunohistochemistry; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D014168:Transferrin / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D004791:Enzyme Inhibitors; D051907:Lysosomal Membrane Proteins; D009097:Multienzyme Complexes; D014168:Transferrin; D005576:Colforsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex