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

Abstract

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.

FIG. 1

Only the C terminus of EF-1β is essential in vivo. (A) Sequence of S. cerevisiae EF-1β indicating sites of truncations (underlined). (B) Western blot analysis of extracts of strains expressing the HA epitope-tagged full-length and truncated yEF-1β. Lanes from left to right are TKY285 (yEF-1β-HA), JWY4298 (yEF-1βΔ60-HA), JWY4299 (yEF-1βΔ85-HA), and TKY257 (yEF-1βΔ96-HA) grown in galactose and expressed from the GAL1 promoter. (C) Growth of strains expressing full-length and truncated yEF-1β. Strains from the top are TKY285 (yEF-1β-HA), JWY4298 (yEF-1βΔ60-HA), JWY4299 (yEF-1βΔ85-HA), and TKY257 (yEF-1βΔ96-HA) grown on YEP-galactose at 30°C for 4 days. (D) Comparison of expression from the GAL1 and TEF5 promoters. Lanes from left to right are TKY285 (GAL1 promoter, yEF-1β-HA) and the TEF5 promoter expressing full-length yEF-1β-HA (TKY258) and truncated yEF-1βΔ96-HA (TKY266). (E) Growth of strains expressing (from top to bottom) TKY285 (GAL1 promoter, yEF-1β-HA) and TEF5 promoter expressing full-length yEF-1β-HA (TKY258) and truncated yEF-1βΔ96-HA (TKY266) grown on YEP-galactose at 30°C for 4 days. All blots were probed with anti-HA and anti-phosphatidylglycerol kinase antibodies. For panels B and D, MW indicates molecular mass in kilodaltons.

FIG. 2

Conserved cluster of residues in S. cerevisiae, S. pombe (fission yeast), X. laevis, B. mori (silkworm), Oryza sativa (rice), A. salina, wheat germ, T. cruzi, rabbit, and human EF-1β proteins and hEF-1δ. Hyphens indicate residues identical to S. cerevisiae EF-1β.

FIG. 3

The human homolog of yEF-1β, but not the hEF-1β-like protein EF-1δ, is functional in yeast. The cDNAs encoding hEF-1β and hEF-1δ were fused to the HA epitope tag and expressed under the control of the yeast GAL1 promoter. (A) Western blot analysis indicates that HA-tagged full-length forms of hEF-1β and hEF-1δ are expressed under the GAL1 promoter. Lanes from left to right are hEF-1δ-HA (JWY4229 with pTKB301), hEF-1β-HA (TKY256), yEF-1β-HA (TKY169), and a strain containing an untagged form of yEF-1β (JWY4229). (B) Truncated hEF-1δ is stably expressed under the control of the GAL1 promoter in yeast. Lanes from left to right are hEF-1δΔ172-HA (JWY4229 with pTKB158) and yEF-1βΔ96-HA (TKY257). (C) Growth of strains containing HA-tagged full-length forms of yEF-1β-HA (TKY169, top) and hEF-1β-HA (TKY256, bottom) on C-Ura-galactose at 30°C for 4 days. (D) Growth of strain JWY4229 expressing full-length and truncated hEF-1δ. The strain contains the chomosomal wild-type TEF5 gene and (from top to bottom) pRS316 (empty vector), hEF-1δ-HA, or hEF-1δΔ172-HA and was grown on C-Ura-galactose at 30°C for 4 days. (E) yEF-1β, hEF-1β, and hEF-1δ coimmunoprecipitate with yEF-1α. Shown are Western blots with anti-HA antibodies of a portion of the supernatants (S) and the entire pellets (P) of an immunoprecipitation of yeast extracts containing yEF-1β-HA (TKY169), hEF-1β-HA (TKY286), and hEF-1δ-HA (TKY4231 plus pTKB151) precipitated with an anti-yEF-1α polyclonal antibody. For panels A, B, and E, MW indicates molecular mass in kilodaltons.

FIG. 4

Growth defects of strains containing the tef5 mutant alleles. (A) Strains containing the lys2-801 allele (UAG) and either the wild-type TEF5 gene or one of the tef5-1 to tef5-10 alleles on a LEU2 CEN plasmid were grown at 30°C, and equal numbers of cells were spotted on YEPD medium. From top left are shown TKY235 (TEF5), TKY238 (tef5-1), TKY240 (tef5-2), TKY244 (tef5-3), TKY236 (tef5-4), TKY242 (tef5-5), TKY251 (tef5-6), TKY243 (tef5-7), TKY239 (tef5-8), TKY241 (tef5-9), and TKY237 (tef5-10). Growth was monitored following 3 to 7 days at 37, 30, 24, or 13°C. (B) Growth was monitored on complete medium–0.5 mg of paromomycin per ml (top) and C-Lys–0.5 mg of paromomycin per ml (bottom) for strains (from top left) TKY235 (TEF5), TKY238 (tef5-1), TKY240 (tef5-2), TKY244 (tef5-3), and TKY243 (tef5-7) following 7 days at 30°C.

FIG. 5

Excess copies of the gene encoding yEF-1α but not of that encoding yEF-1γ (TEF3 and TEF4, respectively) result in a conditional growth defect in a wild-type strain. Strain TKY235 (TEF5 LEU2) was transformed with URA3 plasmids containing no TEF gene (pRS316), TEF2 CEN, TEF2 2μm, TEF3 CEN, and TEF4 CEN. The strains were grown at 30°C in liquid C-Ura, and equal numbers of cells for each were spotted in 1/10 serial dilutions and grown on C-Ura at 13, 24, 30, and 37°C.

FIG. 6

Suppression of the Cs⁻ defects of mutant strains containing tef5-1 (TKY238 S121L) (A) and tef5-4 (TKY236 K120R S121L) (B) by excess yEF-1α. Plasmids bearing the URA3 marker and no TEF gene (pRS316), TEF2 CEN, TEF2 2μm, TEF3 CEN, and TEF4 CEN were transformed into the strain and grown in C-Ura. Equal numbers of cells for each strain were spotted in 1/10 serial dilutions and grown on C-Ura at 30 and 13°C.

FIG. 7

Total methionine incorporation in strains containing wild-type TEF5 (TKY235, circles), the tef5-1 allele (TKY238, squares) (A), or the tef5-7 allele (TKY243, triangles) (B). Strains were grown to mid-log phase in C-Met and labeled for varying times in [³⁵S]methionine. Incorporation (in counts per minute) is expressed per A₆₀₀ unit of cells.