Interactions of elongation factor 1α with F-actin and β-actin mRNA: Implications for anchoring mRNA in cell protrusions

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

Research output: Contribution to journalArticle

129 Scopus citations

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 la (EF1α) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1α colocalizes with β-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and β-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1α in mRNA targeting, we mapped the two actin-binding sites on EF1α 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 EF1α 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 EF1α-F-actin complex is the scaffold that is important for β-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 EF1α and the EF1α-binding site of yeast Bni1p, a protein that inhibits EF1α binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1α or the EF1α-binding site of Bni1p inhibits EF1α binding to β-actin mRNA in vitro and causes delocalization of β-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1α in the anchoring of β-actin mRNA to the protrusion in crawling cells.

Original languageEnglish (US)
Pages (from-to)579-592
Number of pages14
JournalMolecular biology of the cell
Volume13
Issue number2
DOIs
StatePublished - Mar 4 2002
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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