Identification of motifs involved in endoplasmic reticulum retention-forward trafficking of the GLT-1 subtype of glutamate transporter
Identification of motifs involved in endoplasmic reticulum retention-forward trafficking of the GLT-1 subtype of glutamate transporter
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DOI：
10.1523/jneurosci.0839-04.2004
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发表时间：
2004-06-02
影响因子：
5.3
通讯作者：
Robinson, MB
中科院分区：
文献类型：
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作者：
Kalandadze, A;Wu, Y;Robinson, MB
谷氨酸转运蛋白可能以同多聚体形式存在，但对于确保正确组装和表面表达的机制知之甚少。在本研究中，我们研究了谷氨酸转运蛋白 GLT-1 亚型翻译后加工的机制。鉴定出一个基于细胞外亮氨酸的基序，在突变为丙氨酸 (6L/6A GLT-1) 后，会阻止 GLT-1 从内质网 (ER) 输出到质膜，并表现出“不成熟”转运蛋白的糖基化模式特征。该 6L/6A 变体对野生型 GLT-1 表达具有选择性显性失活作用，并与 GLT-1 形成免疫共沉淀复合物。两个下游精氨酸残基突变为丙氨酸部分恢复了 6L/6A 变体的成熟和功能活性。这一额外的突变挽救了 GLT-1 的成熟，这一事实基本上排除了 6L/6A 突变体变体由于简单的错误折叠而未得到适当处理的可能性。当包含这些基序的结构域被引入白细胞介素 2α 受体亚基（Tac 蛋白）中拓扑相似的位置时，突变对蛋白质成熟具有类似的影响。拓扑模型至少将基于亮氨酸的基序放置在细胞外结构域中，该结构域在组装过程中面向内质网的内腔。基于这些数据，我们认为进化上保守的基于精氨酸的基序起到内质网滞留信号的作用，而抑制该信号需要腔内亮氨酸基序。有趣的是，在 CNS 中发现了高比例的缺乏该结构域部分的可变剪接 GLT-1 mRNA，这表明 GLT-1 表达可能在组装过程中受到调节。
Glutamate transporters may exist as homomultimers, but little is known about the mechanisms that ensure proper assembly and surface expression. In the present study, we investigated the mechanisms that contribute to posttranslational processing of the GLT-1 subtype of glutamate transporter. Anextracellular leucine-based motif was identified that after mutation to alanine (6L/6A GLT-1) prevented export of GLT-1 from the endoplasmic reticulum ( ER) to the plasma membrane and displayed a glycosylation pattern characteristic of "immature" transporter. This 6L/6A variant had a selective dominant-negative effect on wild-type GLT-1 expression and formed coimmunoprecipitable complexes with GLT-1. Mutation of two downstream arginine residues to alanine partially restored maturation and functional activity of the 6L/6A variant. The fact that this additional mutation rescued maturation of GLT-1 essentially excludes the possibility that the 6L/6A mutant variant is not appropriately processed because of simple misfolding. When the domain containing these motifs was introduced into a topologically similar location in the interleukin 2alpha receptor subunit (Tac protein), the mutations had a similar effect on protein maturation. Topological models place at least the leucine-based motif in an extracellular domain, which would face the lumen of the ER during assembly. On the basis of these data, we suggest that an evolutionarily conserved arginine-based motif functions as an ER retention signal and a lumenal leucine motif is required for suppression of this signal. Interestingly, a high percentage of variably spliced GLT-1 mRNAs lacking parts of this domain are found in the CNS, suggesting that GLT-1 expression may be regulated during assembly.