The β subunit of the EF-1 complex has demonstrated activity as the guanine nucleotide exchange factor for the G protein EF-1α (6
). For EF-1β as a guanine nucleotide exchange factor, models of G protein regulation allow several predictions of the effects of changes in EF-1β activity. First, although active EF-1α–GTP can be regenerated by the spontaneous release of GDP, EF-1β enhances the rate of this reaction. Thus, EF-1β contributes to a larger pool of active EF-1–GTP, making possible increased rates of translation elongation. Deletion of EF-1β should thus slow elongation, likely below the threshold for viability, consistent with the essential nature of the TEF5
gene encoding yEF-1β (14
). Second, as the substrate for EF-1β, excess EF-1α should be able to at least partially compensate for the loss of EF-1β activity, which we have previously demonstrated for yeast (20
A third prediction is that a critical function catalyzed by EF-1β should be conserved, which we find by both sequence and function conservation. Analysis of the sequence identity among 10 different EF-1β proteins from many different species clearly supports the importance of the C terminus of this protein in its function in vivo. We demonstrate that an EF-1β fragment containing the C terminus is able to function as the only form of the protein in vivo, with no associated growth defects. While previous studies have indicated that a C-terminal protease fragment of A. salina
EF-1β containing residues 106 to 206 maintains guanine nucleotide exchange activity in vitro (40
), these experiments demonstrate that this region is sufficient for normal growth and results in no sensitivity to translation inhibitors or changes in polyribosome content or distribution.
The human homolog of EF-1β is also functional in vivo in yeast. Expression of the hEF-1β-like protein hEF-1δ is unable to complement the lack of yEF-1β in vivo, which may not be surprising since the level of full-length and especially truncated hEF-1δ expressed was lower than that of hEF-1β. It is more surprising that the hEF-1δ was unable to even partially suppress the conditional growth defects of strains containing mutant forms of yEF-1β and actually showed a slight negative effect on cell growth. Since hEF-1δ does strongly physically interact with yEF-1α, as shown by coimmunoprecipitation (Fig. E), this negative growth effect is likely caused by dominantly interfering with the function of yEF-1β. Future analysis of any potential dependence of this association on EF-1β and EF-1γ, with strains deficient in these subunits, will provide insight into the EF-1 complex.
Removal of the more divergent N-terminal region of hEF-1δ negates this effect, likely by reducing the affinity for yEF-1α and relieving inhibition of yEF-1β function. Association of neither hEF-1δΔ172-HA nor yEF-1βΔ96-HA with yEF-1α could be detected by coimmunoprecipitation (data not shown). Thus, the cell tolerates both reduced levels of EF-1β protein, as seen for the truncated forms of yEF-1β or the constructs expressed from the GAL1
promoter (Fig. ), and reduced association with EF-1α, as seen for the truncations (Fig. ), while still allowing normal growth. While hEF-1δ may possess guanine nucleotide exchange activity in vitro and the conservation of sequence allows association with yEF-1α, the small number of changes between hEF-1β and hEF-1δ, which are 85.3% identical in the conserved C-terminal 109 amino acids, are clearly important for EF-1β function. It appears that EF-1δ may be specific to metazoans, perhaps by functioning in the assembly of the higher-order aa-tRNA synthetase complexes found in these organisms (2
), but provides an important tool for analyzing the functional differences between EF-1β and EF-1δ.
Mutations in the conserved C terminus of EF-1β affect both the efficiency and the accuracy of translation elongation. Strains with mutations in residues K120 and S121 of yEF-1β show severe growth defects and sensitivities to elongation inhibitors relative to a wild-type strain. These results indicate that mutations in yEF-1β alter translation elongation, either directly or through the activity of yEF-1α. According to the nuclear magnetic resonance structure of the C terminus of hEF-1β, residues K120 and S121 lie at the end of a β-sheet opposite the loops predicted to play critical roles in guanine nucleotide exchange (28
). Thus, the in vivo analysis of yEF-1β has yielded new insight into residues not predicted to play a critical role in the function of this protein. It is of particular interest that the Cs−
defects of strains containing many of the yEF-1β mutations are suppressed by excess yEF-1α. No effects are seen on the Ts−
mutant phenotype. Cs−
defects are often associated with reduced complex formation; thus, these mutations may reduce the interaction between EF-1α and EF-1β. Alternatively, if EF-1β activity is limiting, the presence of excess EF-1α may allow for growth by a mechanism similar to that seen when EF-1α is overexpressed in cells completely lacking EF-1β (20
). These effects are different from the growth seen when EF-1β activity is totally bypassed by expression of a third copy of an EF-1α gene (20
). EF-1β-deficient strains with three copies of EF-1α are extremely slow growing, while examples such as the tef5-1
allele shown in Fig. are suppressed to wild-type levels of growth.
The critical role of EF-1β in maintaining a pool of active EF-1α–GTP supports the prediction that these mutations would alter translational fidelity. An additional phenotype of strains containing a mutant allele of TEF5
is enhanced sensitivity to paromomycin, which is often predictive of effects on translational fidelity (26
). The lack of readthrough of nonsense mutations in a lacZ
reporter construct and the lys2-801
(UGA) allele clearly demonstrates that mutations in the highly identical K120 and S121 residues enhance the fidelity of nonsense recognition in yeast. Many mutations in tRNA and ribosomal protein genes that reduce translational fidelity and suppress nonsense codons have been isolated (13
). Most mutations that enhance fidelity were isolated and analyzed as antisuppressors of strains bearing suppressor mutations (13
). The effect of the EF-1β mutants is seen for all three nonsense codons, indicating that these mutants show omnipotent hyperaccuracy. This phenotype is similar to the antisuppressor phenotypes that result from overexpression of Sup35p (eRF3) and Sup45p (eRF1) (39
) and some rRNA mutations (8
) in yeast. Thus, a potential mechanism for the increased fidelity at stop codons may be more efficient competition for recognition of the stop codon by release factors due to an increased ratio of the less abundant release factors to active EF-1α.
It is interesting to compare these mutants to the restrictive mutants of bacteria and corresponding mutations in yeast (1
). Restrictive mutants enhance translational fidelity by lowering the stability of the ribosome–aa-tRNA interaction, resulting in an increased aa-tRNA discard rate (22
). The prediction of such mutations is that any increase in translational fidelity would require reduced growth rates and a lower translational efficiency (22
). E. coli
ribosome mutants with altered processivity also show increased accuracy (10
). These predictions are supported by the growth phenotypes and the reduced total translation of strains containing the tef5-1
mutant allele. Thus, EF-1β activity is likely limiting for translation elongation, such that mutations that alter EF-1β activity slow total translation and consequently enhance translational fidelity. Clearly, EF-1β plays an important role in the efficiency and accuracy of the translation elongation process. Further genetic and biochemical analysis of these mutants will provide insight into factors that may regulate translation elongation, such as kinases (7
), or interactions with other components of the translational apparatus such as the ribosome or release factors.