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Vol. 13, Issue 2, 579-592, February 2002
with F-Actin and 
-Actin
mRNA: Implications for Anchoring mRNA in Cell Protrusions
*Department of Anatomy and Structural Biology and
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 1the Analytical Imaging Facility, Albert Einstein College
of Medicine, New York, New York 10461
(EF1) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1 colocalizes with 
-actin mRNA and F-actin in
protrusions of chicken embryo fibroblasts and binds directly to F-actin
and -actin mRNA simultaneously in vitro in actin cosedimentation and
enzyme-linked immunosorbent assays. To investigate the role of EF1
in mRNA targeting, we mapped the two actin-binding sites on EF1 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 EF1 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 EF1-F-actin complex is the
scaffold that is important for -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 EF1 and the EF1-binding site of yeast
Bni1p, a protein that inhibits EF1 binding to F-actin and also is
required for yeast mRNA localization. We demonstrate that either domain
III of EF1 or the EF1-binding site of Bni1p inhibits EF1
binding to -actin mRNA in vitro and causes delocalization of
-actin mRNA in chicken embryo fibroblasts. Taken together, these
results implicate EF1 in the anchoring of -actin mRNA to the
protrusion in crawling cells.
Corresponding author and present address:
Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail address:
liug{at}mail.amc.edu.
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