The Effect of F-actin on the Binding and Hydrolysis of Guanine Nucleotide by Dictyostelium Elongation Factor 1A

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 EF1␣), 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 (K d ) for GDP and GTP are 2.5 M and 0.6 M, respectively, and the k cat of GTP hydrolysis is 1.0 ؋ 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 GTP⅐eEF1A⅐F-actin. We conclude that Factin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP⅐eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.Actin has been implicated as a co-factor in eukaryotic protein synthesis (reviewed in Refs. 1-3). Early cell ultrastructural studies revealed an association between components of the translational machinery and the actin cytoskeleton (4, 5), and functional studies showed that mRNAs associated with detergent-insoluble material, presumably the cytoskeleton, are translated more efficiently (6). Subsequently, it has been shown that virtually all of the protein components of translation associate with the cytoskeleton in situ (7-12); however, no direct effect of the cytoskeleton on the specific activity of any of these co-factors has been demonstrated.Eukaryotic elongation factor 1A (eEF1A, 1 formerly EF1␣) 2 is the most abundant G protein in cells, comprising 1-5% of total cell protein (14 -16). The principal role of eEF1A during protein synthesis is to bind aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism (17). Following the hydrolysis of GTP, aminoacyl-tRNA is incorporated into the growing polypeptide, and GDP⅐eEF1A is released from the ribosome. The exchange of bound GDP for free GTP regenerates the capacity of eEF1A to bind another aminoacyl-tRNA and to participate in further cycles of elongation. Therefore, the affinity of eEF1A for GDP and GTP and the kinetics of GTP hydrolysis are potential targets for ligands operating through eEF1A to regulate protein synthesis. For example, the rate of GDP/ GTP exchange by eEF1A in vitro is too slow to support the measured rate of polypeptide elongation in vivo; therefore, the need for a nucleotide exchange factor has been postulated (18,19).eEF1A was identified in a screen for actin binding proteins in Dictyostelium and was shown to bind both monomeric and filamentous actin (F-actin) in vitro and in situ (20 -23). Due to the striking similarity of eEF1A primary sequences among species, it...

Interactions of Elongation Factor 1α with F-Actin and β-Actin mRNA: Implications for Anchoring mRNA in Cell Protrusions

“…13, February 2002solution (Edmonds et al, 1998). In addition, electron microscopy studies of EF1␣ crossbridges in two-dimensional rafts of actin filaments indicate an interfilament spacing of 120 Å, in agreement with the measured radius of a single globular 50-kDa polypeptide (Edmonds et al, 1999).…”

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

“…Although the eEF1A-actin interaction has been well characterized in vitro (13,40), the consequences of disrupting the interaction in vivo are not fully understood. Overexpression of eEF1A in S. cerevisiae causes a slow growth phenotype and concurrent large cell phenotype arising from disorganization of the actin cytoskeleton (16).…”

Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange

“…Alternatively, this mutation may affect a function of eEF1A not assayed, such as the affinity for the ribosome or actin binding. Guanine nucleotide binding to Dictyostelium eEF1A decreases the affinity for actin 7-7.5-fold (30), and thus the D156N mutant may alter the equilibrium between free and actin-bound eEF1A.…”