Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell
Rabaptin-5-independent membrane targeting and Rab5 activation by Rabex-5 in the cell
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DOI：
10.1091/mbc.e07-02-0100
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发表时间：
2007-10-01
影响因子：
3.3
通讯作者：
Li, Guangpu
中科院分区：
文献类型：
--
作者：
Zhu, Huaiping;Zhu, Guangyu;Li, Guangpu
Rabex-5 是 Rab5 的鸟嘌呤核苷酸交换因子 (GEF)。在这里，我们报告了 Rabex-5 的一个新功能域的鉴定，该功能域对于其膜靶向和 Rab5 GEF 体内活性至关重要。数据显示全长 Rabex-5 有效激活细胞中的 Rab5。然而，GEF 结构域本身（残基 135-399）在这方面是无活性的，尽管其在体外具有活性。一系列 Rabex-5 构建体的生成和表征表明，GEF 结构域无法靶向早期内体，并且 GEF 结构域的 N 端序列可以恢复其早期内体靶向及其在细胞中激活 Rab5 的能力。该区域（残基 81-135）被称为膜结合基序，其与下游螺旋束结构域（残基 135-230）一起形成早期内体靶向（EET）结构域，该结构域对于与早期内体的关联是必要且充分的。此外，一些活性 Rabex-5 构建体的 C 端区域不包含 Rabaptin-5 结合结构域。因此，Rabex-5可以通过EET结构域靶向早期内体，并在体内以不依赖于Rabaptin-5的方式激活Rab5。我们讨论了一个模型，用于将这些体内数据与先前关于 Rabex-5 功能及其与 Rabaptin-5 相互作用的体外结果进行协调。
Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.