Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool that Specifically Traps GTP-Rab35
Genome-wide Investigation of the Rab Binding Activity of RUN Domains: Development of a Novel Tool that Specifically Traps GTP-Rab35
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DOI：
10.1247/csf.11001
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发表时间：
2011-01-01
影响因子：
1.5
通讯作者：
Ohbayashi, Norihiko
中科院分区：
文献类型：
--
作者：
Fukuda, Mitsunori;Kobayashi, Hotaka;Ohbayashi, Norihiko
RUN结构域是由大约70个氨基酸组成的较不保守的蛋白质基序，并且因为RUN结构域经常在参与Rab小GTP酶的调节的蛋白质中发现，所以RUN结构域已经被认为参与Rab介导的膜运输，可能作为Rab结合位点。然而，由于大多数RUN结构域的Rab结合活性从未被研究过，在这项研究中，我们通过酵母双杂交测定法，以60种不同的Rab作为诱饵，对19种不同的含有RUN结构域的蛋白质的RUN结构域的Rab结合活性进行了全基因组分析。结果显示，其中只有6种与具有不同Rab结合特异性的特异性Rab同种型相互作用，即，DEND 5 A/B与Rab 6 A/B，PLEKHM 2与Rab 1 A，RUFY 2/3与Rab 33，RUSC 2与Rab 1/Rab 35/Rab 41。我们还鉴定了RUSC 2的最小功能Rab 35结合位点（氨基酸残基982-1199），并成功开发了一种新的GTP-Rab 35特异性捕获剂，我们将其命名为RBD 35（Rab 35特异性Rab-binding domain specific for Rab 35）。重组RBD 35被发现在体外和PC 12细胞中特异性地捕获GTP-Rab 35，并且在PC 12细胞中过表达荧光标记的RBD 35强烈抑制神经生长因子依赖的神经突生长。
The RUN domain is a less conserved protein motif that consists of approximately 70 amino acids, and because RUN domains are often found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated membrane trafficking, possibly as a Rab-binding site. However, since the Rab binding activity of most RUN domains has never been investigated, in this study we performed a genome-wide analysis of the Rab binding activity of the RUN domains of 19 different RUN domain-containing proteins by yeast two-hybrid assays with 60 different Rabs as bait. The results showed that only six of them interact with specific Rab isoforms with different Rab binding specificity, i.e., DENND5A/B with Rab6A/B, PLEKHM2 with Rab1A, RUFY2/3 with Rab33, and RUSC2 with Rab1/Rab35/Rab41. We also identified the minimal functional Rab35-binding site of RUSC2 (amino acid residues 982-1199) and succeeded in developing a novel GTP-Rab35-specific trapper, which we named RBD35 (Rab-binding domain specific for Rab35). Recombinant RBD35 was found to trap GTP-Rab35 specifically both in vitro and in PC12 cells, and overexpression of fluorescently tagged RBD35 in PC12 cells strongly inhibited nerve growth factor-dependent neurite outgrowth.