title: Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle.
abstract: Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.
authors.affiliation: Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ito
authors.forename: Ichiro
authors.initials: I
authors.lastname: Koshino
authors.forename: Nobuto
authors.initials: N
authors.lastname: Arashiki
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Adachi
authors.forename: Mizuki
authors.initials: M
authors.lastname: Tomihari
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tamahara
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Kurogi
authors.forename: Takashi
authors.initials: T
authors.lastname: Amano
authors.forename: Ken-ichiro
authors.initials: K
authors.lastname: Ono
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Inaba
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013103:Spherocytosis, Hereditary / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex