Abstract
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 eEF1Bα, 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.
THE eukaryotic translation elongation factor 1 (eEF1A, formerly EF-1α) is a highly abundant protein responsible for the delivery of aminoacyl-tRNA (aa-tRNA) to the elongating ribosome. As a prototypical G-protein, the transition between active and inactive forms is based on whether GTP or GDP, respectively, is bound (Bourneet al. 1990). The ribosome acts as a GTPase activator for eEF1A in the presence of a codonanticodon match between the aa-tRNA and the A-site codon of the ribosome-bound mRNA. Correspondingly, eEF1A requires the eEF1B guanine nucleotide exchange factor (GEF) complex, composed of the α-, β-, and γ-subunits (Carvalhoet al. 1984). In the yeast Saccharomyces cerevisiae the eEF1A and eEF1Bα subunits are essential gene products (Nagataet al. 1984; Hiragaet al. 1993). Only the eEF1Bα subunit (formerly EF-1β) is required for nucleotide exchange, since eEF1Bγ (formerly EF-1γ) is not essential (Kambouriset al. 1993; Kinzyet al. 1994) and there is no protein homologous to the metazoan subunit eEF1Bβ (formerly EF-1Δ). Alterations of eEF1 subunit activities, such as mutations that alter either yeast eEF1A and eEF1Bα, affect the efficiency and accuracy of translation in vivo and in vitro (Sandbaken and Culbertson 1988; Dinman and Kinzy 1997; Cavallius and Merrick 1998; Carr-Schmid et al. 1999a,b). Further, a strain with one instead of two copies of the genes encoding eEF1A shows reduced nonsense suppression (Songet al. 1989). The effects of excess eEF1 subunit activity or protein levels are less well understood, and the potential for coordinated expression or feedback regulatory mechanisms such as that found for other components of the translational apparatus, such as ribosomal proteins, is unknown.
eEF1A, like other components of the translation apparatus, has functions in processes aside from translation elongation (reviewed in Kinzy and Goldman 2000). These include effects on viral replication, calmodulin binding, and actin binding and bundling. The ability of eEF1A to bind actin and form unique bundles was demonstrated initially and most extensively in Dictyostelium (Yanget al. 1990). It has been found that eEF1A from many other species also maintains this function (Itano and Hatano 1991; Kurasawaet al. 1996; Sudaet al. 1999). Furthermore, binding to actin is affected by the nucleotide-bound state of eEF1A and is sensitive to cellular parameters such as nucleotide and pH (Edmonds et al. 1995, 1998; Liuet al. 1996). The consequences of the eEF1A-actin interaction in vivo remain unknown, although models link this association to the organization and compartmentalization of the translational apparatus within the cell or cytoskeletal functions independent of translation (reviewed in Condeelis 1995). Further genetic evidence has suggested an interaction through Bni1p, a regulator of actin organization in budding yeast (Umikawaet al. 1998).
Previously, we have demonstrated that overexpression of the TEF2-encoded eEF1A protein results in slow growth of S. cerevisiae (Carr-Schmidet al. 1999b). We have expanded our understanding of the consequences of elongation factor overexpression for both forms of eEF1A as well as the catalytic subunit of eEF1B, eEF1Bα. The overexpression of either protein results in slow and conditional growth defects. These effects, however, are not a result of general effects on translation elongation, as measured by translation rates and sensitivity to translation inhibitors. Neither are these effects due to altered reading frame maintenance or readthrough of stop codons. To dissect other likely causes of this effect, alterations in actin localization and function were assessed. Consistent with an interaction of eEF1A with actin in vivo, overexpression of eEF1A affects actin distribution, morphology, and budding. However, neither eEF1Bα overexpression nor strains with a mutant form of eEF1A affecting nucleotide binding and fidelity show this effect. To further understand the role of this interaction in vivo, we examined a potential genetic interaction between actin and eEF1A. Utilizing a bank of actin mutants, we have demonstrated a synthetic relationship between a subset of actin alleles and increased eEF1A levels. This suggests that there is a genetic interaction between actin and eEF1A, providing additional in vivo evidence that the two proteins functionally interact. We show, through biochemical methods, that eEF1A from S. cerevisiae, like that from other organisms, is a bona fide actin binding and bundling protein. Thus, biochemical data support the genetic interactions between the two proteins and the resulting effects on the cell.
MATERIALS AND METHODS
Strains and media: Escherichia coli DH5 was used for plasmid work. S. cerevisiae strains are shown in Table 1. Standard yeast genetic methods were employed (Shermanet al. 1986). Yeast cells were grown in either YEPD (1% Bacto yeast extract, 2% peptone, 2% dextrose) or defined synthetic complete media (C or C–) supplemented with 2% dextrose or 2% galactose as a carbon source. Yeast were transformed by the lithium acetate method (Itoet al. 1983).
DNA manipulations: Restriction endonucleases and DNA modifying enzymes were obtained from Boehringer Mannheim Biochemicals (Indianapolis). The low-copy (CEN) vectors pRS314 (TRP1), pRS315 (LEU2), and pRS316 (URA3) and the high-copy vectors pRS424 (TRP1), pRS425 (LEU2), and pRS426 (URA3) were used (Sikorski and Hieter 1989; Christiansonet al. 1992). The URA3-based TEF2 vectors for low-copy (YCpMS29) and high-copy (YEpMS42) expression and the URA3-based TEF5 vector for low-copy expression (pTKB269) were previously described (Sandbaken and Culbertson 1988; Carr-Schmidet al. 1999b). pTKB168 (TEF1 CEN LEU2) was prepared by subcloning an EcoRV fragment containing TEF1 from pJS7 (Songet al. 1989) into pBluescript (pJWB3014) and removing the TEF1 gene as an ApaI SmaI fragment into pRS315. pTKB274 (TEF1 2μ LEU2) was prepared by subcloning the EcoRV fragment containing TEF1 from pJWB3014 into pRS424. pTKB316 (TEF5-HA 2μ URA3) was prepared by digesting pTKB269 with EagI and XhoI and subcloning into pRS426. pTKB247 (TEF5 CEN TRP1) and pTKB226 (TEF5 2μ TRP1) were prepared by cloning the ClaI fragment containing TEF5 from pJWB2953 into pRS314 and pRS424. pTKB478 (TEF5 CEN URA3) and pTKB479 (TEF5 2μ URA3) were prepared by digesting pJWB3013 with BamHI and XhoI and subcloning into pRS316 and pRS426, respectively.
Western blot analysis: Yeast strain TKY259 containing either the vectors pRS314 and pRS315, pRS314 and pTKB168 (TEF1 CEN), or pRS314 and pTKB274 (TEF1 2μ) for the TEF1 overexpression series; vectors pRS314 and pRS316, pRS314 and YCpMS29 (TEF2 CEN), or pRS314 and YEpMS42 (TEF2 2μ) for the TEF2 overexpression series; vectors pRS315 and pRS316, pRS315 and pTKB269 (TEF5-HA CEN), or pRS315 and pTKB316 (TEF5-HA 2μ) for the TEF5-HA overexpression series; or vectors pRS315 and pRS316, pRS315 and pTKB478 (TEF5 CEN), or pRS315 and pTKB479 (TEF5 2μ) for the untagged TEF5 overexpression series were maintained in selective double drop-out medium. Cells were grown in liquid C-Leu-Trp (TEF1 series), C-Ura-Trp (TEF2 series), or C-Ura-Leu (TEF5-HA and TEF5 series) to an A600 in mid-log phase (∼0.6 to 1.0 units) and extracts prepared by glass bead lysis. Approximately 0.5 μg of total protein, as determined by Bradford protein analysis (Bio-Rad, Hercules, CA), were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with polyclonal antibodies to yeast eEF1A (1:5000 dilution), Rpa1p (1:1000 dilution, kindly provided by Dr. Steven Brill, Rutgers University), or yeast eEF1Bα (1:1000 dilution), or a monoclonal antibody to the hemagglutinin (HA) epitope tag on eEF1Bα (1:100 dilution) and detected for multiple time points by a secondary antibody conjugated to alkaline phosphatase (1:10,000 dilution, Bio-Rad) or peroxidase (1:10,000 dilution, Amersham ECL) to ensure a linear response.
Sensitivity of yeast strains to temperature, drugs, and actin or eEF1A overexpression: Temperature (Ts–), cold (Cs–), and drug sensitivity were assayed for the strains described above on appropriate double drop-out medium essentially as described in Carr-Schmid et al. (1999a). The wild-type (IGY191) and act1 mutant strains were transformed with the 2μ vectors Yep24 (URA3) or YEpMS42 (URA3 TEF2). Cells at an A600 of 1.0 were spotted on C– medium lacking the corresponding amino acids followed by incubation at 13°,23°, 30°, and 37° for the overexpression series or 18°,23°, 30°,34°, and 37° for the actin mutant series for 3–7 days. For drug sensitivity, 0.3 ml of culture was spread plated onto selective media plates and 10 μl of either 250 mm paromomycin or 0.5 or 1 mm cycloheximide were pipetted onto sterile BBL ¼-in.-diameter paper discs and incubated for 2–3 days at 30°. The ability of eEF1A and eEF1Bα overexpression to suppress the lethality of actin overexpression was assayed in strain IGY191 transformed with pTKB168, pTKB274, or pJWY3013 and either empty pRS316 or pAAB218, expressing ACT1 from a GAL1 promoter. Cells were streaked on C-Ura-Leu and C-Ura-Leu + galactose and grown for 3–6 days at 30°.
Nonsense suppression assays: Nonsense suppression assays were performed on strain TKY259 containing pRS314 and pRS315, TEF1 (pTKB168 or pTKB274), and pRS315, untagged TEF5 (pTKB247 or pTKB226), and pRS314 or all pairwise combinations of the TEF1 and TEF5 plasmids. All nine strains were transformed with the URA3 wild-type lacZ control plasmid pUKC815tail (lacZ under the PGK1 promoter with the PGK1 transcriptional terminator) or URA3 plasmids with in-frame nonsense codons in lacZ; pUKC819tail (UGA), pUKC817tail (UAA), and pUKC818tail (UAG; Carr-Schmidet al. 1999b). The mutant and wild-type strains containing each plasmid were grown overnight at 30° in C-Ura-Leu-Trp to mid-log phase. At least four samples for each strain and reporter plasmid were analyzed in duplicate using the ONPG assay previously described (Dinman and Kinzy 1997).
S. cerevisiae strains
In vivo [35S]methionine incorporation: Strain TKY259 containing pRS315 and pRS314, pRS314 and pTKB168 (TEF1 CEN), pRS314 and pTKB274 (TEF1 2μ), pRS315 and pTKB247 (TEF5 CEN), or pRS315 and pTKB226 (TEF5 2μ) were grown in liquid cultures (100 ml) in C-Met-Trp-Leu at 30° to an A600 of 0.5–0.7 and assayed as described in Carr-Schmidet al. 1999b). All time points were analyzed in triplicate.
Protein purification: Wild-type actin was purified from AAY1453 by the method of Honts et al. (1994) with the following revisions: the concentration of Tris-HCl in the lysis and column buffers was increased to 20 mm and G-actin was eluted from the DE-52 column with a single 300 mm KCl wash. eEF1A was purified by the method of Cavallius et al. (1997) with the following adjustments. Cell pellets were flash frozen in liquid nitrogen, stored at –80° until ready for use, thawed, and washed in 20 mm Tris-HCl pH 7.5 prior to resuspension in lysis buffer. All solutions for lysis and elution are as previously described except that they contained 0.25 mm GDP and 25% glycerol. Cell lysis was carried out for 1 hr by cycling between 30 sec “on” and 90 sec “off” in a Bead Beater (Biospec Products, Bartlesville, OK). Prior to mixing the cell lysate with the DEAE-cellulose, the lysate was adjusted to 100 mm KCl. The unbound material from the diethylaminoethyl cellulose (DEAE)-cellulose (DE-52, Whatman) batch column was mixed with phosphocellulose (P-11, Whatman) and washed with elution buffer containing 100 mm KCl until the wash solution had an A280 of 0. Elution from the phosphocellulose column and loading and elution from the CM-cellulose (CM-52, Whatman) column were as previously described. Fractions containing >95% pure eEF1A were identified by SDS-PAGE.
Actin-eEF1A binding and bundling assays: Binding and bundling assays were performed by adaptations of procedures previously described (Liuet al. 1996). Prior to use in these assays, the eEF1A was dialyzed into cosedimentation assay buffer (20 mm PIPES, pH 7.2, 2 mm EGTA, 1 mm DTT, 1 mm ATP, 2 mm MgCl2, 1 mm PMSF, and 0.25 mm GDP) for 4 hr at 4°. Dialyzed eEF1A and purified G-actin were clarified by centrifugation at 130,000 × g in a Beckman TLA-100 tabletop ultracentrifuge for 40 min at 4°. For all procedures, the Beckman polycarbonate tubes were treated with Sigmacote, rinsed with cosedimentation assay buffer, and dried prior to use. G-actin (5 μm) was added to cosedimentation assay buffer, followed by the addition of 1 μm eEF1A to give a total volume of 100 μl. This mixture was incubated for 18–20 hr at 4° to allow equilibration. The mixture was then divided: 50 μl was centrifuged at low speed (50,000 × g; 36,000 rpm in Beckman TLA-100) for 2 min at 4° to assay bundling and 50 μl was centrifuged at high speed (130,000 × g; 60,000 rpm in Beckman TLA-100) for 40 min at 4° to assay binding. Supernatants and pellets were separated and solubilized in SDS-PAGE sample buffer.
Electron microscopy: Prior to centrifugation, samples of the mixtures prepared for low-spin cross-linking assays were placed into wells. Briefly, carbon-coated mica was dipped into each sample allowing the carbon to partially lift off the mica and collect the sample. Carbon-coated samples were negatively stained in 2% uranyl acetate, 125 mm NaCl, 22.5 mm HEPES pH 7.5, 1 mm MgCl2, and 2 mm EGTA by the same method and transferred completely off the mica onto sterile water and collected on 300-mesh grids (Valentineet al. 1968). Samples were viewed using a JEOL 100cx microscope at 80 kV. The identity of each sample remained unknown until after scoring.
Phalloidin staining, quantitation of budding defects, and flow cytometry analysis: Strain TKY259 containing either pRS316, excess eEF1A (YCpMS29 or YEpMS42), or excess eEF1Bα (pTKB478 or pTKB479) was grown in minimal medium supplemented with 0.25% casamino acids, tryptophan, and adenine for ∼36 hr, maintaining log phase by continual dilution. To enhance staining, 90 min prior to fixation, the cell cultures were pelleted and resuspended in an equal volume of YEPD. Fixation and staining were performed by the method of Chowdhury et al. (1992) using rhodamine phalloidin (Molecular Probes, Eugene, OR). A sample of a mid-log phase culture grown at 30° in C-Ura was analyzed for the percentage of budded and unbudded cells by sonicating a sample for 6 sec followed by counting 500 cells and determining the relative size of the bud to the mother. Flow cytometry was performed essentially as described in Nash et al. (1988) on strain TKY259 containing either pRS316 and pRS314, excess eEF1A (YCpMS29 or YEpMS42 and pRS314), or excess eEF1Bα (pTKB247 or pTKB226 and pRS316). Briefly, 1 × 107 cells were cultured at mid-log phase in C-Ura-Trp, washed once, sonicated, and resuspended in 1 ml of 50 mm sodium citrate pH 7.0. The cells were then treated with 0.25 mg/ml of Rnase A at 50° for 1 hr, washed, and resuspended in 1 ml 50 mm sodium citrate. Propidium iodide was added to 16 μg/ml and incubated at room temperature for 30 min and the cells were sonicated and analyzed on a Beckman Epics-profile II.
RESULTS
Overexpression of either the TEF1 or TEF2 gene affects cell growth: Previously, during an analysis of the ability of overexpression of the eEF1A protein to suppress the phenotypic defects of cells with mutations in the GEF eEF1Bα, we noted the negative consequences of overexpression of eEF1A expressed from the TEF2 gene (Carr-Schmidet al. 1999b). We determined if expression of the other gene encoding eEF1A, TEF1, showed similar effects. Low (CEN)- and high (2μ)-based plasmids expressing TEF1 were prepared and transformed into strain TKY259, which contains the wild-type complement of the two genes encoding eEF1A (TEF1 and TEF2). Overexpression of either TEF1 or TEF2 on a 2μ plasmid results in slower growth at all temperatures tested (13°, 30°, and 37°, Figure 1, A and B). To confirm that the eEF1A protein is overexpressed, Western blot analysis was performed on cells overexpressing either TEF1 (Figure 1C) or TEF2 (Figure 1D). An extra copy of either gene results in a slight increase in the level of the eEF1A protein when present on a CEN plasmid and a more significant increase when present on a 2μ plasmid.
Overexpression of TEF5 (eEF1Bα) shows a slight effect on cell growth: The eEF1A protein is restored to an active GTP-bound state by the activity of a GEF, eEF1B. In the yeast S. cerevisiae, only the eEF1Bα subunit is required (Kinzyet al. 1994). Previously, we had shown that overexpression of the nonessential eEF1Bγ subunit of the GEF does not affect cell growth (Carr-Schmidet al. 1999b). An analysis of extra copies of the catalytic subunit eEF1Bα encoded by the TEF5 gene is shown in Figure 2. The experiment was performed using both untagged and an HA-epitope-tagged form of the plasmid-encoded TEF5 gene. Comparison of the overexpression effect of eEF1A and eEF1Bα indicates that the presence of a low- or high-copy untagged eEF1Bα-expressing plasmid also results in measurable effects on cell growth (Figure 2A). The eEF1Bα-HA protein is functional in vivo as the only form of the protein (Carr-Schmidet al. 1999b); however, CEN or 2μ plasmids encoding the HA-tagged protein show no effect on cell growth (data not shown). Western blot analysis to monitor the level of the plasmid-encoded proteins shows that either the eEF1Bα-HA protein or untagged protein is expressed at increasing levels in these strains (Figure 2, B and C). Additionally, overexpression of eEF1Bα does not affect the level of expression of eEF1A (Figure 2, B and C), and overexpression of eEF1A does not affect eEF1Bα levels (data not shown).
—Overexpression of eEF1A from either the TEF1 or TEF2 gene affects cell growth. Strain TKY259 containing the wild-type complement of two genes encoding eEF1A (TEF1 and TEF2) was transformed with either A or C: the vectors pRS314 and pRS315 (No), pRS314 and pTKB168 (TEF1 CEN), or pRS314 and pTKB274 (TEF1 2μ) or B and D: the vectors pRS314 and pRS316 (No), pRS314 and YCpMS29 (TEF2 CEN), or pRS314 and YEpMS42 (TEF2 2μ). Cells were grown in C-Leu-Trp (TEF1 overexpression) or C-Ura-Trp (TEF2 overexpression) to mid-log phase at 30°. In A and B cells were diluted to an A600 of 1.0 and spotted as 10-fold serial dilutions on C-Leu-Trp or C-Ura-Trp, respectively, and incubated at 37°, 30°, or 13° for 3 to 7 days. In C and D total proteins were extracted and equal amounts of protein, as determined by Bradford assay, were run on a Laemmli gel, transferred to nitrocellulose, and probed with polyclonal antibodies to Rpa1p (as a loading standard, top arrow) and yeast eEF1A (bottom arrow).
Attempts were made to co-overexpress both eEF1A and eEF1Bα. These experiments proved difficult, however, in that cells overexpressing both proteins reverted from extreme slow growth to growth similar to that seen with overexpression of one subunit. It is likely in these cases that the cells select for colonies maintaining lower copy numbers of the 2μ plasmids. Western blot analysis within days of transformation shows that both proteins are overexpressed; however, the level decreases with time between transformation and analysis. In general, however, the level of eEF1A tends to reach the level found when eEF1A alone is present on a CEN plasmid and eEF1Bα showed a dosage-dependent increase (data not shown).
—Overexpression of eEF1Bα affects cell growth. Strain TKY259 containing the wild-type complement of one gene encoding eEF1Bα (TEF5) was transformed with the vectors pRS315 and pRS316 (No) and either A and B: untagged eEF1Bα (pRS315 and pTKB478 [TEF5 CEN]) or pRS315 and pTKB479 (TEF5 2μ) or in C: HA-tagged eEF1Bα (pRS315 and pTKB269 [TEF5-HA CEN] or pRS315 and pTKB316 [TEF5-HA 2μ]). (A) Cells overexpressing untagged eEF1Bα were diluted to an A600 of 1.0 and spotted as 10-fold serial dilutions on C-Leu-Ura and incubated at 37°, 30°, or 13°, for 3–7 days. (B and C) Cells were grown in C-Leu-Ura to mid-log phase at 30°. Total proteins were extracted and equal amounts of protein, as determined by Bradford assay, were run on a Laemmli gel, transferred to nitrocellulose, and probed with polyclonal antibodies to Rpa1p (as a loading standard, top arrow), yeast eEF1A (middle arrow), and (B) a polyclonal antibody to yeast eEF1Bα or (C) a monoclonal antibody to the HA epitope tag on eEF1Bα (bottom arrow).
Overexpression of either eEF1A or eEF1B1α shows no effect on protein synthesis: To determine the mechanism of the negative effect of elongation factor over-expression on cell growth, several monitors of the translational status of the cell were utilized. Strains overexpressing either eEF1A or eEF1Bα were assayed for altered sensitivity to the translation inhibitor cycloheximide and paromomycin; however, no change was seen (data not shown). Strains with or without overexpression of eEF1A from a CEN (pTKB168, Figure 3A, open circles) or 2μ (pTKB274, Figure 3A, solid circles) plasmid or eEF1Bα from a CEN (pTKB247, Figure 3B, open squares) or 2μ (pTKB226, Figure 3B, solid squares) plasmid were assayed for changes in total translation by [35S]methionine labeling. No significant effect on total translation was seen with overexpression of either eEF1A or eEF1Bα.
Since general effects on translation were not seen, more subtle effects on translational fidelity were monitored. Cells overexpressing either or both factors were assayed for the ability to allow retrotransposition of the yeast Ty1 element, a process dependent on a programmed +1 ribosomal frameshift (Farabaugh 1995). No strain where either eEF1A, eEF1Bα, or both proteins were overexpressed showed any alteration in the frequency of retrotransposition (data not shown). Additionally, effects on the ability to accurately read the three stop codons were monitored by β-galactosidasebased reporter constructs. Singularly, eEF1A and eEF1Bα affected nonsense suppression to a minor extent at most (Table 2). Co-overexpression of both eEF1A and eEF1Bα increased nonsense suppression significantly at all three stop codons (Table 2), in particular when eEF1Bα was present on a 2μ plasmid.
—Strains overexpressing either eEF1A or eEF1Bα show no effect on total protein synthesis. Strain TKY259 expressing no additional elongation factors (pRS314 and pRS315, triangles) and A: with excess eEF1A; TEF1 CEN (pRS314 and pTKB168, open circles) or TEF1 2μ (pRS314 and pTKB274, solid circles) or B: with excess eEF1Bα TEF5 CEN (pRS315 and pTKB247, open squares) or TEF5 2μ (pRS315 and pTKB226, solid squares) were grown in C-Met-Trp-Leu at 30° to an A600 of 0.5–0.7. Cold methionine (50 μm) and 1 μCi/ml [35S]methionine (7.9 mCi/ml, 293.0 MBq/ml, New England Nuclear, Boston) were added to each culture and both the A600 and cold trichloroacetic acid precipitable radioactivity were determined at each time point. The graphs express the cpm/A600 at the indicated time point and represent the average of at least four data points.
Previously, we showed that mutations in the NXKD GTP-binding consensus element of eEF1A that result in altered translational fidelity do not affect cell growth (Carr-Schmidet al. 1999a). Because those mutants result in a greater decrease in accuracy than cells overexpressing eEF1A (above), the growth defects associated with overexpression of eEF1A cannot be explained just by altered translational fidelity. The studies above therefore suggest that a function other than its role in translation is affecting cell growth. Numerous studies of various cell types have indicated that eEF1A can bind to actin in vitro, and it therefore seemed possible that overexpression of eEF1A in yeast might thereby affect the actin cytoskeleton. We tested this hypothesis by asking whether (i) the distribution of actin and cell morphology was altered in cells overexpressing eEF1A; (ii) actin mutants show genetic interactions with overproduction of eEF1A; and (iii) eEF1A and actin from yeast, like those from other cell types, interact in vitro.
Cells overexpressing eEF1A, but not eEF1Bα, show altered actin localization, cell morphology, and cell cycle distribution: The effect of overexpression of eEF1A and eEF1Bα on cell morphology and cell cycle distribution was examined by light microscopy and flow cytometry. Five hundred cells of each overexpression strain were counted to determine the distributions of budded and unbudded cells (Table 3). Excess eEF1A, but not eEF1Bα, increased the fraction of unbudded cells. In addition, overexpression of eEF1A frequently resulted in larger and rounder cells. To confirm this result, flow cytometry was performed on cells overexpressing either eEF1A or eEF1Bα. Overexpression of eEF1A on a 2μ plasmid resulted in an increased representation of cells in the 1N peak (Figure 4).
Since overexpression of eEF1A resulted in altered effects on the cell, we tested the possibility that the ratio of actin to eEF1A is important in maintaining normal cell homeostasis. In S. cerevisiae, overexpression of eEF1A results in slow growth (above) and expression of actin from a GAL1 promoter is lethal (Liuet al. 1992). In Schizosaccharomyces pombe, overexpression of eEF1A from the thiamine-inducible promoter is lethal while overexpression of actin has little to no effect but suppresses the lethality of eEF1A overexpression (Sudaet al. 1999). We tested the possibility that overexpression of actin and eEF1A or eEF1Bα could suppress each other's defects in S. cerevisiae. Overexpression of neither eEF1A (CEN or 2μ) nor eEF1Bα (CEN) was able to even partially suppress the lethality of actin overexpression (data not shown).
Since actin in yeast has effects on cell morphology and budding, effects on the distribution of actin were analyzed by staining cells with fluorescent-labeled phalloidin. The cells overexpressing eEF1A from a 2μ plasmid are large, and actin is distributed along the length of the buds (Figure 5A). Normally, cells show a concentration of actin at the tip of the bud (Botsteinet al. 1997). In contrast, cells overexpressing eEF1A from a CEN plasmid or cells overexpressing eEF1Bα showed normal morphology and actin distribution similar to wild-type cells with a control plasmid. Mutations in eEF1A that affect nucleotide affinity and fidelity (Carr-Schmidet al. 1999a) do not show effects on actin distribution and cell morphology (Figure 5B).
eEF1A overexpression causes synthetic growth defects of the actin mutants: To further test for in vivo interactions, we determined whether eEF1A and actin interact genetically by examining the effect of overexpression of eEF1A on wild-type and act1 mutant strains. We reasoned that overexpression of eEF1A in the actin mutant collection might suppress or exacerbate the actin mutant phenotype.
Nonsense suppression of strains overexpressing eEF1A or eEF1Bα
We overexpressed eEF1A by introducing a high-copy (2μ) plasmid containing either TEF2 or no insert into wild-type and various otherwise isogenic act1 mutant strains. Because the actin mutants show a variety of cold-sensitive and temperature-sensitive phenotypes, we tested growth of each strain at five temperatures: 18°, 23°, 30°, 35°, and 37° (Table 4). The wild-type strain (IGY191) showed reduced growth in the presence of extra copies of eEF1A at 30°,34°, and 37°. This defect is similar to that discussed for the wild-type strain TKY259 above, and, except in the case of IGY191, there were no growth defects detected at temperatures <30°. Many of the mutants showed moderate to significant reduction in growth at high temperature, which may be an additive effect of overexpressing eEF1A in cells that are already sick. Three of the mutants, act1-2, act1-120, and act1-125, showed a major reduction in growth at low temperature (18°, Figure 6). Not all actin mutant strains show this effect (Figure 6, act1-119 and act 1-124 and data not shown). This reduction is particularly significant, as the otherwise isogenic wild-type strain does not show any reduction in growth at this temperature in the presence of overexpressed eEF1A (Figure 6), and at least in the cases of act1-120 and act1-125 the cells grow well at this temperature in the absence of extra copies of eEF1A. This finding suggests that the defects due to mutations in actin and overexpressed eEF1A are synthetic and not merely additive. Interestingly, however, several actin mutants, act1-116, act1-117, act1-122, and act1-123, appear resistant to the negative effect of overexpressing eEF1A at most temperatures. Overexpression of the eEF1A protein in the wild-type and mutant strains was essentially as observed in TKY259 (data not shown), and no suppression of the growth defect of any actin mutant was observed.
Budding patterns of cells overexpressing eEF1A or eEF1Bα
Yeast eEF1A binds and cross-links yeast actin in vitro: The studies above suggested that a plausible explanation for the growth defect of cells overexpressing eEF1A is that the eEF1A-actin interaction is being affected by the excess eEF1A, resulting in altered distributions of actin, altered cell morphology, and exacerbated defects of actin mutants. As it was not known whether actin and eEF1A from yeast, like those from other cell types (Yanget al. 1990; Itano and Hatano 1991; Kurasawaet al. 1992; Sudaet al. 1999), interact with each other physically, we conducted biochemical experiments to examine the interaction between these two proteins. Binding of purified yeast eEF1A to yeast actin filaments was assessed by cosedimentation. Actin alone (Figure 7A), eEF1A alone (Figure 7B), or eEF1A and actin (Figure 7C) were mixed, incubated for 18–24 hr under conditions that promote actin polymerization, and subjected to high speed centrifugation. High speed centrifugation is sufficient to pellet the individual actin filaments, but not the eEF1A (Figure 7, A and B). However, in the presence of actin filaments, eEF1A is found primarily in the pellet along with the actin filaments (Figure 7C). This indicates that eEF1A binds to the actin filaments. Cross-linking of yeast actin filaments by yeast eEF1A was assessed by low speed centrifugation. At this speed, individual actin filaments and individual molecules of eEF1A do not sediment (Figure 7, A and B). However, in the presence of eEF1A, the actin filaments and associated eEF1A do sediment (Figure 7C), indicating that eEF1A cross-links the actin filaments.
—Strains overexpressing eEF1A show an increased representation in G1. Strain TKY259 with no additional elongation factors (NO, pRS316 and pRS314), excess eEF1A encoded by TEF2; CEN (YCpMS29 and pRS314) or 2μ (YEpMS42 and pRS314) or excess eEF1Bα; CEN (pTKB247 and pRS316) or 2μ (pTKB226 and pRS314) were grown to mid-log phase in C-Ura-Trp, stained with propidium iodine, and analyzed by flow cytometry.
Electron microscopy was used to visualize the individual actin filaments (Figure 8A) and actin filaments cross-linked by eEF1A (Figure 8B). In the absence of eEF1A, actin is clearly present as individual filaments, while in the presence of eEF1A, actin is clearly cross-linked into bundles. The cross-linked actin filaments appear to have square-packed bundles, similar to those observed in other organisms (Owenet al. 1992). Taken together, these data establish that S. cerevisiae eEF1A is a bona fide yeast actin filament cross-linking protein, similar to that observed in other organisms.
—Strains overexpressing eEF1A show altered actin distribution, morphology, and budding. (A) Strain TKY259 with no additional elongation factors (wild type, pRS316), excess eEF1A; TEF2 CEN (YCpMS29) or TEF2 2μ (YEpMS42) or excess eEF1Bα; TEF5 CEN (pTKB478) or TEF5 2μ (pTKB479) were analyzed. (B) Wild-type (MC213) and eEF1A mutants TKY278 (D156N) and TKY280 (N153T) were analyzed. Cells were grown at 30° to an A600 of 0.5–0.7, stained with phalloidin, and photographed at 100× magnification.
DISCUSSION
The two genes for eEF1A encode identical amino acid sequences, and eEF1A from either gene can be overexpressed with resulting effects on cell growth. Additionally, eEF1Bα can also be overexpressed and, to a lesser extent, overexpression affects cell growth. Thus, neither subunit appears subject to the feedback regulation seen in other components of the translational apparatus, such as ribosomal proteins (reviewed in Woolford and Warner 1991). In addition, neither eEF1 subunit affects the expression of the other, since eEF1A protein levels are unchanged when eEF1Bα is overexpressed and vice versa. Interestingly, the HA-epitope-tagged form of eEF1Bα does not induce a growth defect even though it is efficiently overexpressed. The HA-tagged protein is functional in vivo as the only form of the protein, and a strain expressing the tagged protein shows no defects on cell growth or general assays of protein synthesis (Carr-Schmidet al. 1999b). Therefore, it appears likely that the N terminus may play a role in the function causing the eEF1Bα overexpression phenotype. This result is also consistent with our findings that mutant forms of eEF1Bα are stably made but are not functional when tagged at the N terminus (L. Valente and T. G. Kinzy, unpublished observations). Thus, while not essential (Carr-Schmidet al. 1999b), the N terminus is likely involved in cellular functions of eEF1Bα. Previously, we have shown that overexpression of either form of eEF1Bγ, encoded by TEF3 or TEF4, does not have an effect on cell growth (Carr-Schmidet al. 1999b). Recently, however, TEF4 overexpression was linked to altered translational fidelity (Benkoet al. 2000). We have shown that mutant forms of eEF1A that alter nonsense suppression up to fourfold show no effect on growth (Carr-Schmidet al. 1999a). It is perhaps not surprising that larger effects on fidelity are required before growth defects are observed, and the growth defects observed with eEF1 subunit overexpression likely result from alterations in other cellular functions.
Growth at various temperatures of the act1 alleles containing extra copies of TEF2 encoded eEF1A
The observation that overexpression of eEF1A causes a growth defect in S. cerevisiae could at first glance be easily interpreted in light of the normally tight regulation of protein synthesis and the possibility that excess eEF1A could result in altered rates of elongation or compete improperly with release factors for termination codons. The phenotype of eEF1Bα overexpression could be interpreted similarly by increasing the pool of active eEF1A. However, using multiple assays for effects on total translation and reading frame maintenance, no effects of overexpression of either protein on protein synthesis are seen. Minor effects on nonsense suppression begin to appear with eEF1A or eEF1Bα overexpression individually, but are more pronounced with co-overexpression of the two proteins. The near lethal phenotype of high level expression of both proteins, however, results in a high rate of reversion and lower expression of eEF1A. Thus, the growth defects of these strains, which are slightly more dramatic for eEF1A than for eEF1Bα overexpression, may be the result of other cellular errors. During co-overexpression the effect of eEF1Bα appears to manifest more in a nonsense suppression phenotype, especially when eEF1A is slightly overexpressed, indicating a potential link to A-site misreading or termination.
—Overexpression of eEF1A in some actin mutants reduces growth at low temperatures. Strains 191 (ACT1), IGY54 (act1-120), IGY61 (act1-125), IGY116 (act1-2), IGY51 (act1-119), and IGY56 (act1-124) containing either the wild-type or a mutant allele of the ACT1 gene were transformed with the vector YEp24 (top) or YEpMS42 (URA3 TEF2 2μ, bottom). Cells were grown in C-Ura, diluted to an A600 of 1.0, spotted as 10-fold serial dilutions on C-Ura, and incubated at 18° for 7 days.
An obvious target for altered function in the presence of excess eEF1A is actin. eEF1A and actin from numerous organisms have been shown to interact in vitro (reviewed in Condeelis 1995), and it therefore seemed likely that overexpression of eEF1A might result in an altered intracellular distribution of actin. To test this possibility, we examined the intracellular distribution of actin in cells overexpressing eEF1A and found that the localization of actin was altered in these cells. In yeast, actin has a role in budding and the polarization of growth (Kilmartin and Adams 1984). We therefore examined the ability of cells overexpressing eEF1A to bud and form normal-shaped cells. We found that overexpression of eEF1A on a 2μ plasmid resulted in defective budding and enlarged cells, while no such effects are seen for eEF1Bα overexpression. These observations suggest that overexpression of eEF1A may directly affect the actin cytoskeleton and that the growth defects seen in cells overexpressing eEF1A may be a consequence of this change. These findings also raise the possibility that a subset of mRNAs may be more sensitive to eEF1A overexpression and the resulting alteration in the actin cytoskeleton, because mammalian protein synthesis requires an intact F-actin system (Stapulioniset al. 1997). Recent reports have shown that eEF1A overexpression in S. pombe can affect morphology and actin distribution (Sudaet al. 1999); however, the results are distinctly different from S. cerevisiae. Overexpression of eEF1A is lethal in S. pombe while actin overexpression is lethal in S. cerevisiae. While overexpression of actin suppresses the eEF1A lethality in S. pombe, co-overexpression of the two proteins in S. cerevisiae shows no such relationship.
—S. cerevisiae eEF1A binds and cross-links actin. SDS-PAGE of low speed supernatants (LSS), low speed pellets (LSP), high speed supernatants (HSS), and high speed pellets (HSP) obtained by centrifugation of A, purified yeast actin, B, purified yeast eEF1A, or C, purified actin polymerized in the presence of eEF1A as described in materials and methods. The positions of yeast actin (A) and yeast eEF1A (EF) protein bands in the gels are indicated. The proteins were resolved in a 10% SDS-polyacrylamide gel and stained with Coomassie blue R-250.
—S. cerevisiae eEF1A stimulates formation of actin bundles. Electron microscopy of actin filaments in the absence (A) and presence (B) of eEF1A. Samples of the cross-linking assays were examined by negative staining using electron microscopy as described in materials and methods. Bar, 1 μm.
To further test for an in vivo interaction between actin and eEF1A, we asked whether actin and eEF1A interact with each other genetically. In particular, we tested the effects of overexpression of eEF1A in a large collection of otherwise isogenic actin mutant strains. We found that overexpression of eEF1A resulted in a synthetic growth defect in some, but not all, mutant strains. The act1-2, act1-120, and act1-125 alleles are particularly sensitive to overexpression of eEF1A. These are not the sickest of the mutant collection, indicating it is not simply synthetic with slow growth. Furthermore, it is of interest that act1-120 and act-125 remove negative charges, which could potentially interact with the very basic eEF1A protein, and these residues are surface exposed in both G- and F-actin (Figure 9). It is also possible, however, that the effect is not directly through eEF1A, but that these actin alleles may be more sensitive to changes in the translational apparatus and the link between the cytosketeton and efficient protein synthesis (Stapulioniset al. 1997). Finally, it remains to be determined if eEF1A binding and bundling competes with other actin binding proteins. Although the molecular basis for the eEF1A-actin interaction is unknown at present, we show that in S. cerevisiae, the biochemical interaction between eEF1A and actin is conserved. These results suggest that actin and eEF1A interact with each other in vivo and make it likely that the observed effects of eEF1A overexpression on the actin cytoskeleton are functionally significant. Further mapping of the allele specificity of these and related phenotypes with the available crystal structures of the two proteins may help lead to a better understanding of the nature and location of this interaction. Overexpression of S. cerevisiae eEF1A was recently identified as a suppressor on a synthetically lethal arc1 los1 mutant strain (Grosshanset al. 2000). Since Los1p, the Xpo-t homolog in yeast, and Arc1p, an aminoacylation cofactor, are linked to tRNA pools, the results indicated that eEF1A plays a role in tRNA export and that reduced levels or activity of eEF1A alter export. It is possible that should the balance of eEF1A available for protein synthesis, actin binding, and tRNA export be upset, multiple negative consequences may occur. The ability to alter cell growth by altering eEF1A levels without dramatically affecting translation establishes a system to further characterize the multiple roles of eEF1A. These results support the link between eEF1A and the cytoskeleton with likely effects on related processes such as budding.
—The mutated side chains of act1 alleles synthetic with overexpression of eEF1A map to surface-accessible residues on a single side of the molecule. Side chain substitutions in act1-2, act1-120, and act1-125 are shown and reside in areas containing C-alpha atoms more than 12 Å away from any C-alpha atoms in the neighboring molecule in F-actin (light color). Coordinates from pdb 1YAG were superimposed on the model of F-actin (pdb 1ALM) using the program MOLMOL (Koradiet al. 1996).
Acknowledgments
The authors thank members of the Kinzy and Adams laboratories and Nancy Walworth for helpful discussions and Gregers Rom Andersen for comments on the manuscript and Figure 9. This work was supported by U.S. Public Health Service grants GM57483 (to T. G. Kinzy), GM45288 (to A. E. M. Adams), and a Cancer Biology Training Grant (CA09213) to J. L. Whitacre and K. A. Kandl.
Footnotes
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Communicating editor: Fred Winston
- Received August 31, 2000.
- Accepted December 4, 2000.
- Copyright © 2001 by the Genetics Society of America
