Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle
Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle
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DOI：
10.1242/jcs.03101
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发表时间：
2006-09-01
影响因子：
4
通讯作者：
Inaba, Mutsumi
中科院分区：
文献类型：
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作者：
Ito, Daisuke;Koshino, Ichiro;Inaba, Mutsumi
AE 1（阴离子交换器1，带3）基因的各种突变导致显性遗传性球形红细胞增多症，这是一种常见的先天性溶血性贫血，与不同程度的AE 1缺乏和红细胞膜突变蛋白丢失有关。为了确定AE 1蛋白水平降低的机制，我们采用K562和HEK 293细胞系和非洲爪蟾卵母细胞与牛野生型AE 1和R664 X无义突变体，负责显性遗传性球形红细胞增多症，分析蛋白表达，营业额和细胞内定位。R664 X-突变蛋白经历了快速降解，并导致特别增加营业额和受损的运输到质膜的野生型蛋白通过异源寡聚体形成在K562细胞。与这些观察结果一致，突变体和野生型AE 1的共表达减少了卵母细胞中野生型蛋白的阴离子转运。在K562和HEK 293细胞中的转染研究表明，介导R664 X和野生型AE 1降解的主要途径通过蛋白酶体途径采用内质网（ER）相关降解。R664 X蛋白的蛋白酶体降解似乎独立于泛素化和N-糖基化，并且在蛋白酶体抑制后未观察到侵略体形成。这些发现表明与显性遗传性球形红细胞增多症相关的AE 1 R664 X蛋白对野生型AE 1的表达具有显性负效应。
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 wildtype 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.