The Saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation factor

Abstract

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated Mr 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.

FIG. 1

Analysis of the protein and the mRNA encoded by the TIF6 gene. (A) Expression of yeast eIF6 in E. coli. Cell extracts, prepared from IPTG-induced cultures of E. coli BL21(DE3) cells harboring either the parental plasmid pET-5a (lane 1) or pET-TIF6 (lane 2), were electrophoresed in an SDS–15% polyacrylamide gel and subjected to Coomassie blue staining. The position of migration of the expressed protein of about 26 kDa is shown by an arrowhead. (B) Immunoblot analysis of the bacterially expressed 26-kDa protein with mammalian anti-eIF6 antibodies as probes. Cell extracts of IPTG-induced cultures of BL21(DE3) cells harboring either the parental vector (lane 1) or the pET-TIF6 recombinant expression plasmid (lane 2) were subjected to Western blot analysis with monospecific anti-mammalian eIF6 antibodies (32) as probes. (C) Immunoblot analysis with anti-TIF6p (eIF6) antibodies as probes. The protein samples used in lanes 1 and 2 were the same as those used in lanes 1 and 2 of panel B, while lane 3 contained 100 μg of a partially fractionated protein fraction derived from cell extracts of S. cerevisiae W303 (a/α). Immunoblot analysis was carried out with anti-yeast 26-kDa Tif6p antibodies as probes. A set of molecular weight marker proteins was run in a separate lane of each gel (data not shown). The position of eIF6 is shown by an arrowhead. (D) Northern blot analysis of mRNA expressed from the TIF6 gene. A Northern blot containing 2 μg of electrophoretically separated yeast poly(A)⁺ RNA was hybridized to a ³²P-labeled 735-bp TIF6 ORF. The blot also contained a set of RNA size markers, of which the position of 1.35-kb RNA is indicated. The blot was analyzed by autoradiography.

FIG. 2

Ribosomal-subunit anti-association activity of bacterially expressed recombinant yeast eIF6. eIF6 activity was measured by the ability of the protein to prevent the association of 40S and 60S ribosomal subunits at 5 mM Mg²⁺ to form 80S ribosomes, as described previously (37). Three reaction mixtures, each of 100 μl and containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 1 mM dithiothreitol, and 1.0 A₂₆₀ unit of Artemia salina 80S ribosomes, were prepared. Two of the reaction mixtures, A and B, contained no protein factors, while mixture C contained 1.25 μg of purified recombinant yeast eIF6 (Sephadex G-75 fraction). After incubation at 30°C for 5 min, the Mg²⁺ concentration of mixtures B and C was raised to 5 mM while that of mixture A was kept at 1 mM, and the incubation was continued for another 5 min at 37°C. Each reaction mixture was then chilled in an ice-water bath, loaded onto a 5-ml linear 5 to 30% (wt/vol) sucrose gradient containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM dithiothreitol, and 5 mM MgCl₂ (for mixtures B and C) or 1 mM MgCl₂ (for mixture A), and centrifuged for 90 min in an SW50.1 rotor at 48,000 rpm. Each gradient was fractionated and the A₂₅₄ profile was analyzed by using an UA-5 absorbance monitor. (A) No yeast eIF6 added, reaction at 1 mM Mg²⁺; (B) no yeast eIF6 added, reaction at 5 mM Mg²⁺; (C) yeast eIF6 added, reaction at 5 mM Mg²⁺.

FIG. 3

Analysis of eIF6 depletion in yeast cells and its effect on cell growth. (A) Schematic representation of the plasmid pUB-TIF6. The plasmid was constructed as described under Materials and Methods. Abbreviations: UAS, the upstream activation sequence of the GAL10 promoter; Ub, ubiquitin gene; X, the codon for arginine; lacI, a restriction fragment that encodes amino acid residues 318 to 346 of the lac repressor; HA, an epitope from the influenza virus hemagglutinin protein. (B) Exponentially growing cultures of W303α (○) and KSY603 (•) were diluted to an A₆₀₀ of about 0.03 in either YPGal (galactose) medium or YPD (glucose) medium. Cell growth was monitored by measuring the A₆₀₀. To keep cultures in exponential growth, they were diluted in fresh medium whenever the A₆₀₀ reached 0.8 U. (C) At the indicated times following the shift from YPGal to either YPGal (left) or YPD (right), cell lysates were prepared from KSY603 as described in Materials and Methods. Approximately 50 μg of protein from each cell lysate was electrophoresed through an SDS–15% polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane. The blot was then probed with peroxidase-coupled anti-HA monoclonal antibodies to detect eIF6 fusion protein. The decrease in the amount of eIF6 observed at the 6-h time point in YPGal is due to a gel loading error.

FIG. 4

Inhibition of protein synthesis in eIF6-depleted cells. Exponentially growing cultures of W303α or KSY603 growing in galactose medium lacking methionine (SGal-Met medium) were harvested, and approximately 10 A₂₆₀ units of cells was suspended in 150 ml of either SD-Met (glucose) or SGal-Met medium and grown at 30°C. At the indicated times, 1 A₂₆₀ unit of cells from each culture was harvested and suspended in 300 μl of either SD-Met or SGal-Met medium containing 50 μCi of [³⁵S]methionine (1175 Ci/mmol). (A) Protein synthesis rates were determined as described in Materials and Methods. The rate of protein synthesis at each time point was calculated as counts of ³⁵S radioactivity incorporated per microgram of protein per minute. (B) Each yeast strain, as indicated, were pulse-labeled for 5 min with [³⁵S]methionine in SD-Met medium, chased for 3 min with 1.5 mM nonradioactive methionine, and lysed. Similar amounts of total protein were separated by SDS-PAGE and autoradiographed.

FIG. 5

Analysis of the polyribosome profiles of eIF6-depleted yeast cells. Exponentially growing cultures of KSY603 or W303α in YPGal medium were harvested and resuspended in either glucose (YPD) or YPGal medium such that the initial A₆₀₀ of each culture was about 0.04 U. At about 5 h (E) and 10 h (B, C, and F) after the shift, 50 ml of each culture was treated with 50 μg of cycloheximide per ml and chilled cells were harvested, washed, and lysed as described in Materials and Methods. About 10 A₂₆₀ units of each cell lysate was subjected to 7 to 47% (wt/vol) sucrose gradient centrifugation. Each gradient was fractionated in an ISCO gradient fractionator, and the A₂₅₄ profile was analyzed in an ISCO UA-5 absorbance monitor. The positions of the half-mer polysomes are indicated by arrowheads (▾) in panels E and F.

FIG. 6

eIF6 depletion results in a decrease in 60S subunits levels. Total ribosomes were isolated from strains W303α and KSY603 after 10 h of growth in either YPD or YPGal, dissociated into 40S and 60S ribosomal subunits, and sedimented through 15 to 40% sucrose gradients as described in Materials and Methods.

FIG. 7

In vitro translation of total yeast poly(A)⁺ RNA in eIF6-depleted cell extracts. (A) Exponentially growing cultures of KSY603 or W303α cells in YPGal medium were harvested and suspended in glucose (YPD) medium such that the initial A₆₀₀ was about 0.03 U. At about 11 h after the shift, the cells were harvested and translation cell extracts were prepared, incubated, and analyzed for [³⁵S]methionine incorporation into proteins as described in Materials and Methods. Where indicated, 5 μg of total yeast poly(A)⁺ RNA (mRNA) and 0.5 μg of recombinant yeast eIF6 were added to 50 μl of reaction mixtures containing 25 μCi of [³⁵S]methionine and all the other components required for translation including micrococcal nuclease-treated cell extracts containing approximately 150 μg of proteins. Aliquots (8 μl) from 50-μl reaction mixtures were withdrawn at the indicated times and analyzed for [³⁵S]methionine incorporation into proteins as described in Materials and Methods. (B) At the indicated times following the shift from YPGal to YPD medium, cell lysates were prepared and 200 μg of protein from each lysate was subjected to Western blot analysis with either anti-HA monoclonal antibodies or anti-L3 antibodies as probes. It should be noted that a relatively large amount of cell lysates was analyzed in the Western blot to ensure that the lysates had no detectable levels of eIF6. Under these conditions, the level of the 60S ribosomal protein L3 was also decreased. However, no change in the level of L3 in cell lysates between 0 and 11 h was apparent in the immunoblot analysis, presumably because the amount of L3 in cell lysates analyzed was still large.

FIG. 8

Translation of luciferase mRNA in cell extracts. Translation cell extracts were prepared from W303α (TIF6) or KSY603 (GAL10::UbTIF6) cells grown in glucose-containing medium for 11 h as described in Materials and Methods. Where indicated, 2 μg of capped luciferase mRNA was added to 50-μl reaction mixtures along with all the other components necessary for in vitro translation. The reactions were carried out at 25°C. At the indicated times, an aliquot of each reaction mixture was assayed for luciferase activity as described by Russel et al. (25).

FIG. 9

Association of eIF6 with 60S ribosomal subunits in yeast cells. An exponentially growing culture of KSY603 was harvested, the cells were washed and lysed, and the cell lysate was subjected to 5 to 30% sucrose gradient centrifugation to separate polysomes, free 80S ribosomes, and ribosomal subunits as described in the legend to Fig. 5 and Materials and Methods. Fractions from the gradient were collected, and proteins were precipitated with 10% trichloroacetic acid, analyzed by SDS-PAGE, and immunoblotted with either anti-L3 antibodies or anti-HA antibodies as probes. Fractions containing 40S and 60S subunits, 80S monosomes, and polysomes are indicated. Protein L3 was used as a marker for 60S subunit sedimentation.