Zhu Huaiping, Zhu Guangyu, Liu Jay, Liang Zhimin, Zhang Xuejun C, Li Guangpu
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Mol Biol Cell. 2007 Oct;18(10):4119-28. doi: 10.1091/mbc.e07-02-0100. Epub 2007 Aug 15.
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.
Rabex-5是Rab5的鸟嘌呤核苷酸交换因子（GEF）。在此，我们报告了Rabex-5一个新功能域的鉴定，该功能域对其在体内的膜靶向作用和Rab5 GEF活性至关重要。数据表明全长Rabex-5能在细胞中有效激活Rab5。然而，GEF结构域本身（第135 - 399位氨基酸残基）在这方面无活性，尽管其在体外具有活性。一系列Rabex-5构建体的生成及特性分析表明，GEF结构域无法靶向早期内体，而GEF结构域N端的一个序列可恢复其早期内体靶向能力及其在细胞中激活Rab5的能力。该区域（第81 - 135位氨基酸残基）被称为膜结合基序，它与下游的螺旋束结构域（第135 - 230位氨基酸残基）共同形成了一个早期内体靶向（EET）结构域，该结构域对于与早期内体的结合是必需且充分的。此外，几个有活性的Rabex-5构建体在C端区域不包含Rabaptin-5结合结构域。因此，Rabex-5可通过EET结构域靶向早期内体，并在体内以不依赖Rabaptin-5的方式激活Rab5。我们讨论了一个模型，以协调这些体内数据与先前关于Rabex-5功能及其与Rabaptin-5相互作用的体外研究结果。
