Overexpression of translation elongation factor 1A affects the organization and function of the actin cytoskeleton in yeast.

The translation elongation factor 1 complex (eEF1) plays a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome. The eEF1A subunit, a classic G-protein, also performs roles aside from protein synthesis. The overexpression of either eEF1A or eEF1B alpha, the catalytic subunit of the guanine nucleotide exchange factor, in Saccharomyces cerevisiae results in effects on cell growth. Here we demonstrate that overexpression of either factor does not affect the levels of the other subunit or the rate or accuracy of protein synthesis. Instead, the major effects in vivo appear to be at the level of cell morphology and budding. eEF1A overexpression results in dosage-dependent reduced budding and altered actin distribution and cellular morphology. In addition, the effects of excess eEF1A in actin mutant strains show synthetic growth defects, establishing a genetic connection between the two proteins. As the ability of eEF1A to bind and bundle actin is conserved in yeast, these results link the established ability of eEF1A to bind and bundle actin in vitro with nontranslational roles for the protein in vivo.

Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange.

A series of mutations in the highly conserved N(153)KMD(156)GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro. All three mutant proteins also show an increase in the K(m) for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1Balpha (eEF1Balpha) and supported for growth by excess eEF1A was used to show the two mutations with the highest K(m) for GTP restore most but not all growth defects found in these eEF1Balpha deficient-strains to near wild type. An increase in K(m) alone, however, is not sufficient for suppression and may indicate eEF1Balpha performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo.

Mutations in elongation factor 1beta, a guanine nucleotide exchange factor, enhance translational fidelity.

Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth. This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved. Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity.