Abstract
Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1alpha), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 10(-3) s-1. When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.
Highlights
Actin has been implicated as a co-factor in eukaryotic protein synthesis
Following the hydrolysis of GTP, aminoacyl-tRNA is incorporated into the growing polypeptide, and GDP1⁄7eEF1A is released from the ribosome
Observations that stimulation of many cell types induces an increase in the rate of protein synthesis and a redistribution of specific mRNAs and eEF1A to the filamentous actin (F-actin)-rich leading edge of lamellipods suggest a link between the protein synthetic activity of eEF1A and its novel bundling of actin filaments at the leading edge [16, 26, 28, 29]
Summary
Actin has been implicated as a co-factor in eukaryotic protein synthesis (reviewed in Refs. 1–3). Dictyostelium actin (2.0 M) was co-polymerized with varying concentrations of eEF1A for 18 h at 22 °C in the above nucleotide-binding buffer containing 1 mM GDP or GTP.
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