Interactions of elongation factor 1α with F-actin and β-actin mRNA:: Implications for anchoring mRNA in cell protrusions
Interactions of elongation factor 1α with F-actin and β-actin mRNA:: Implications for anchoring mRNA in cell protrusions
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DOI：
10.1091/mbc.01-03-0140
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发表时间：
2002-02-01
影响因子：
3.3
通讯作者：
Condeelis, J
中科院分区：
文献类型：
--
作者：
Liu, G;Grant, WM;Condeelis, J
mRNA和局部蛋白质合成的靶向对于细胞极性的产生和维持是重要的。作为翻译机器的一部分以及肌动蛋白/微管结合蛋白，延伸因子1 α（EF 1 α）是蛋白质翻译装置和细胞骨架之间的候选接头。在这项工作中，我们证明EF 1alpha共定位与β-肌动蛋白mRNA和F-肌动蛋白在鸡胚成纤维细胞的突起，并直接结合到F-肌动蛋白和β-肌动蛋白mRNA同时在体外肌动蛋白共沉降和酶联免疫吸附试验。为了研究EF 1 α在mRNA靶向中的作用，我们以高分辨率绘制了EF 1 α上的两个肌动蛋白结合位点，并在结构域I的N端49个残基和结构域III的C端54个残基处定义了一个位点。在体外肌动蛋白结合试验和重组全长EF 1 α及其各种截断体在体内的定位表明，结构域III的C末端是在体外和体内的优势肌动蛋白结合位点。我们认为EF 1 α-F-肌动蛋白复合物是β-肌动蛋白mRNA锚定的重要支架。这种复合物的破坏将导致mRNA的离域。这一假设进行了测试，通过使用两个显性负的多肽：肌动蛋白结合结构域III的EF 1 α和EF 1 α结合位点的酵母Bni 1 p，一种蛋白质，抑制EF 1 α结合F-肌动蛋白，也需要酵母mRNA定位。我们证明，无论是结构域III的EF 1alpha或EF 1alpha结合位点的Bni 1 p抑制EF 1alpha结合β-肌动蛋白mRNA在体外，并导致离域β-肌动蛋白mRNA在鸡胚成纤维细胞。综上所述，这些结果暗示EF 1 α在爬行细胞中将β-肌动蛋白mRNA锚定在突起上。
The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/ microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.