Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRΔF508
Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRΔF508
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DOI：
10.1091/mbc.e07-06-0601
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发表时间：
2008-04-01
影响因子：
3.3
通讯作者：
Nagata, Kazuhiro
中科院分区：
文献类型：
--
作者：
Morito, Daisuke;Hirao, Kazuyoshi;Nagata, Kazuhiro
内质网(ER)中错误折叠或错误组装的蛋白质被输出到胞浆中，并通过泛素-蛋白酶体途径降解，这一过程被称为内质网相关降解(ERAD)。酿酒酵母Hrd1p/Der3p是一种跨越ER膜的泛素连接酶，当囊性纤维化跨膜电导调节因子(CFTR)在酵母细胞中外源表达时，它参与CFTR的ERAD。酵母Hrd1p/Der3p的两个哺乳动物同源基因gp78和Hrd1已被报道。在这里，我们证明了gp78，而不是Hrd1，通过特异性地促进CFTR Delta F508的泛素化，参与了CFTR突变体CFTR Delta F508的ERAD。结构域交换实验和缺失分析表明，gp78通过泛素结合区与CFTR Delta F508结合，即所谓的泛素到内质网降解(CUE)结构域的偶联。Gp78在体外可与N端泛素-谷胱甘肽-S转移酶融合蛋白发生泛素化反应，但不能单独作用于GST。这表明gp78识别已经与CFTR Delta F508结合的泛素，并以类似于多泛素链组装因子(E4)的方式催化CFTR Delta F508的进一步多泛素化。此外，我们通过小干扰RNA方法揭示了泛素连接酶RMA1作为gp78上游的E3酶的功能。我们的数据表明，gp78与RMA1在CFTR Delta F508的ERAD中具有E4样活性。
Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR Delta F508, by specifically promoting ubiquitylation of CFTR Delta F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR Delta F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR Delta F508 and catalyzes further polyubiquitylation of CFTR Delta F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR Delta F508.