Mutational analysis reveals potential phosphorylation sites in eukaryotic elongation factor 1A that are important for its activity

Abstract

Previous studies have suggested that phosphorylation of translation elongation factor 1A (eEF1A) can alter its function, and large-scale phospho-proteomic analyses in Saccharomyces cerevisiae have identified 14 eEF1A residues phosphorylated under various conditions. Here, a series of eEF1A mutations at these proposed sites were created and the effects on eEF1A activity were analyzed. The eEF1A-S53D and eEF1A-T430D phosphomimetic mutant strains were inviable, while corresponding alanine mutants survived but displayed defects in growth and protein synthesis. The activity of an eEF1A-S289D mutant was significantly reduced in the absence of the guanine nucleotide exchange factor eEF1Bα and could be restored by an exchange-deficient form of the protein, suggesting that eEF1Bα promotes eEF1A activity by a mechanism other than nucleotide exchange. Our data show that several of the phosphorylation sites identified by high-throughput analysis are critical for eEF1A function.

Keywords: elongation factor; guanine nucleotide exchange factor; phosphorylation; translation; yeast.

Fig. 1

Localization of proposed phosphorylation sites analyzed on the crystal structure of Saccharomyces cerevisiae eEF1A bound to a C‐terminal fragment of eEF1Bα (PDB 1IJF; [55]). (A) Conserved, potential phosphorylation sites (red) are found in all three domains of eEF1A (blue). (B) Position of residue S289 (magenta) of eEF1A (blue) is shown in relation to eEF1Bα (yellow). Mg²⁺ (green dot) GDP (gray mesh).

Fig. 2

Cells expressing eEF1A‐S289D are temperature and salt‐sensitive. Strains expressing WT (TKY1717) or S289A (TKY1719) or S289D (TKY1720) mutant forms of eEF1A were grown to an A ₆₀₀ of 3 in YEPD. 10‐fold serial dilutions were spotted onto YEPD media (top two panels) or YEPD containing 0.8 m NaCl (bottom panel). Plates were incubated at 30 °C or 37 °C as indicated for 2 days.

Fig. 3

eEF1A‐S289D show protein synthesis defects in vitro but not in vivo. (A) Saccharomyces cerevisiae strains expressing (TKY1717) or the S53A (TKY1718), T82D (TKY1733), S289D (TKY1720) and T430A (TKY1731) mutant forms of eEF1A were grown to log phase in C‐Met at 30 °C. ³⁵[S] methionine was added, and total protein synthesis was measured by TCA precipitation at the indicated time points. Incorporation (counts per min) is expressed per A ₆₀₀ unit. Error bars represent standard error. (B) Wild type and mutant eEF1A from strains as in figure were purified from yeast and assayed in a polyU directed polyphenylalanine synthesis assay. The amount of polyphenylalanine produced in each reaction was measured following TCA precipitation. The activity of the mutant proteins is expressed as a percentage of the amount of polyphenylalanine synthesized by WT eEF1A. Error bars represent standard error.

Fig. 4

The interaction between eEF1A‐S289D and eEF1Bα is significantly reduced. Cell lysates from strains expressing either WT or S289 mutant forms of eEF1A as in Fig. 2 were incubated with control IgG (lanes 5–7) or and a polyclonal antibody to eEF1A (lanes 2–4). Immunoprecipitates were run on SDS/PAGE gels and immunoblotted with antiserum to eEF1Bα (upper panel) or eEF1A (lower panel). Input (lanes 8–10) represents 4% of the cell lysate used in the immunoprecipitation.

Fig. 5

The function of eEF1A‐S289D is compromised in the absence of eEF1Bα encoded by TEF5. (A) Schematic representation of the plasmid shuffle experiment in which overexpression of eEF1A compensates for the loss of eEF1Bα. (B) The tef5Δ strains lacking eEF1Bα and overexpressing a form of eEF1A from plasmids pTKB929 (eEF1A‐WT), pTKB1216 (eEF1A‐S53A), pTKB1218 (eEF1A‐S289A), or pTKB1219 (eEF1A‐S289D) were grown to an A ₆₀₀ of 3 in YEPD. 10‐fold serial dilutions of the indicated strains were pinned on YEPD plates and grown at 30 °C for 3 days. (C) The strains utilized in B. were transformed with either an empty vector, a plasmid expressing WT eEF1Bα, or a plasmid expressing the nucleotide exchange‐deficient mutant, eEF1Bα‐K205A and maintained on C‐Trp‐Leu medium. Pinning was performed as in B, and plates were grown for 2 days at 30 °C.