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延伸因子1α与F-肌动蛋白和β-肌动蛋白mRNA的相互作用:对mRNA锚定在细胞突起中的意义。

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

作者信息

Liu Gang, Grant Wayne M, Persky Daniel, Latham Vaughan M, Singer Robert H, Condeelis John

机构信息

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.

出版信息

Mol Biol Cell. 2002 Feb;13(2):579-92. doi: 10.1091/mbc.01-03-0140.

Abstract

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.

摘要

信使核糖核酸(mRNA)的靶向作用和局部蛋白质合成对于细胞极性的产生和维持至关重要。作为翻译机制的一部分以及一种肌动蛋白/微管结合蛋白,延伸因子1α(EF1α)是蛋白质翻译装置与细胞骨架之间的一个候选连接物。我们在这项研究中证明,EF1α与鸡胚成纤维细胞突起中的β-肌动蛋白mRNA和F-肌动蛋白共定位,并且在肌动蛋白沉降和酶联免疫吸附测定中,它在体外能同时直接结合F-肌动蛋白和β-肌动蛋白mRNA。为了研究EF1α在mRNA靶向中的作用,我们以高分辨率绘制了EF1α上的两个肌动蛋白结合位点,确定一个位点在结构域I的N端49个残基处,另一个位点在结构域III的C端54个残基处。体外肌动蛋白结合测定以及重组全长EF1α及其各种截短体在体内的定位表明,结构域III的C端在体外和体内都是主要的肌动蛋白结合位点。我们提出,EF1α-F-肌动蛋白复合物是对β-肌动蛋白mRNA锚定很重要的支架。该复合物的破坏会导致mRNA的定位缺失。我们用两种显性负性多肽对这一假说进行了验证:EF1α的肌动蛋白结合结构域III和酵母Bni1p的EF1α结合位点,Bni1p是一种抑制EF1α与F-肌动蛋白结合的蛋白质,也是酵母mRNA定位所必需的。我们证明,EF1α的结构域III或Bni1p的EF1α结合位点在体外均抑制EF1α与β-肌动蛋白mRNA的结合,并导致鸡胚成纤维细胞中β-肌动蛋白mRNA的定位缺失。综上所述,这些结果表明EF1α参与了β-肌动蛋白mRNA在爬行细胞中向突起的锚定过程。

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