Eukaryotic translation initiation factor 4E-dependent translation is not essential for survival of starved yeast cells

Abstract

The eukaryotic translation initiation factor 4E (eIF4E) interacts with the mRNA 5' cap structure (m(7)GpppX) and is essential for the appropriate translation of the vast majority of eukaryotic mRNAs. Most studies of the yeast Saccharomyces cerevisiae CDC33 gene product, eIF4E, have been carried out with logarithmically growing cells, and little is known about its role in starved, nonproliferating cells that enter the stationary phase (SP). It has previously been found that the rate of translation in SP cells is more than 2 orders of magnitude lower than it is in dividing yeast cells. Here we show that this low rate of translation is essential for maintaining the viability of starved yeast cells that enter SP. Specifically, starved cells whose eIF4A is inactive or treated with cycloheximide rapidly lose viability. Moreover, after heat inactivation of the cdc33 temperature-sensitive product, the synthesis of most proteins is abolished and only a small group of proteins is still produced. Unexpectedly, starved cdc33 mutant cells whose eIF4E is inactive and which therefore fail to synthesize the bulk of their proteins remain viable for long periods of time, indistinguishable from their isogenic wild-type counterparts. Taken together, our results indicate that eIF4E-independent translation is necessary and sufficient for survival of yeast cells during long periods of starvation.

FIG. 1

The eIF4E level decreases following the shift from log phase to SP. Wild-type (WT) cells and their isogenic cdc33-42 counterparts (see Materials and Methods) were grown in YPD at 25°C. Samples of equal amounts of cells were harvested at either mid-logarithmic growth phase (1 × 10⁷ cells/ml) (lane Log), 24 h later when cells had just entered SP (1.5 × 10⁸ cells/ml) (lane SP0), or 9 days later (1.5 × 10⁸ cells/ml) (lane SP9). Proteins were extracted, and 100-μg samples were analyzed by Western analysis with anti-eIF4E antibodies that recognize one major band at the expected molecular weight, as detailed in Materials and Methods. Equal loadings were verified by ponsau S staining (not shown) and by the presence of a faint band corresponding to a 40-kDa protein that cross-reacted with the anti-eIF4E antibodies (marked by an arrow). Overexposure was required to detect the cross-reactive protein. (A) Analysis of eIF4E extracted from the wild type. (B) Analysis of mutant eIF4E extracted from cdc33–42 mutant cells. (C) Quantitative analysis. One hundred micrograms of protein (left lanes) or an equal volume of the indicated dilutions (right lanes) was analyzed by the Western blot technique as described for panels A and B except that in this experiment the film was exposed for various lengths of time. Shown is the longest exposure time used to detect the faint band in lane SP9. The values indicated at the bottom of panels A and B are calculations of the fold reduction in the level of eIF4E in SP cells compared with its level in log-phase cells. The values in panel A were determined by scanning the film with an ImageScanner (Amersham Pharmacia Biotech) and using the ImageMaster 1D prime program (Pharmacia). The values in panel B were based on the data shown in panel C. For calculating the fold reduction, the film was scanned and the final results were based on both the fold dilution and results obtained by the scanner (whose linear range is limited).

FIG. 2

The level of the cdc33–42 product declines when growing or starved nongrowing cells are exposed to 34°C. Wild-type (WT) cells and their isogenic cdc33–42 counterparts were grown in YPD at 25°C. At mid-logarithmic growth phase (1 × 10⁷ cells/ml), cell samples were taken and the rest of the culture continued to grow to SP. The log-phase samples were either incubated at 25°C or shifted to 34°C. Four hours later, equal numbers of cells were harvested and their proteins were extracted. One day after cells had entered SP, the procedure was repeated. Samples (100 μg) were analyzed by Western analysis as described in the legend to Fig. 1. Equal loadings were verified as described for Fig. 1.

FIG. 3

Inactivation of eIF4E in both growing and starved cells results in substantial translational inhibition. (A to D) Wild-type (WT) cells and their isogenic cdc33–42 counterparts were grown in YPD at 25°C to mid-logarithmic growth phase (A and B) or until 12 h after they entered SP (C and D), when half of each sample was rapidly shifted to 34°C while the other half was left at 25°C. Cell samples were shaken at the indicated temperatures for an additional 4 h, and then each sample was treated as follows. (A and B) Cells (5 × 10⁷) were collected by centrifugation, resuspended in 0.5 ml of the supernatant, and immediately incubated at either of the indicated temperatures. For determining incorporation kinetics, 50 μl of a cell sample was taken, put in an Eppendorf tube, and incubated at the indicated temperatures for 5 min. Radioactive material was then added, and incorporation kinetics were determined as described in Materials and Methods. (C and D) Samples (50 μl) of cells in SP were put in an Eppendorf tube and incubated for 5 min at the indicated temperatures and further treated as described for panels A and B. (E) Wild-type cells and their isogenic cdc33–42 counterparts were grown in synthetic complete medium lacking methionine to SP. One day after entering SP, the culture was shifted to 34°C and shaken for an additional 4 h. Samples (50 μl) of cells were put in an Eppendorf tube and incubated for 5 min at 34°C and further treated as described for panels A and B, except that twice as much radioactive material was added. Note that, since the medium lacked methionine, the specific activity of the radioactive methionine was much higher than it was in the experiments whose results are shown in panels A to D. In the inset, wild-type cells and their isogenic cdc33–42 counterparts were grown in synthetic complete medium lacking methionine. Three days after cells entered SP, the culture was shifted to 34°C and shaken for an additional 16 h. Samples (500 μl) of cells were labeled for 1 h at 34°C with 300 μCi/ml. Because protein degradation in SP is repressed (13, 21), labeling of proteins for 1 h reflects mainly, if not exclusively, their synthesis rates (21). Proteins were extracted as detailed in Materials and Methods, and 10 μg of protein was loaded per lane, electrophoresed, and fluorographed as described in Materials and Methods. Note the different scales of the y axes of the various panels.

FIG. 4

During SP, eIF4A-mediated and cycloheximide-sensitive translation is essential for cell viability but inactivation of eIF4E has no effect on viability. Wild-type (WT) cells or the indicated isogenic mutant cells were grown in YPD at 25°C. In mid-logarithmic growth phase (1 × 10⁷ cells/ml) (A and C) or after cells had entered the SP (B and D), the culture was shifted to the nonpermissive temperature. Cultures used in the experiments whose results are shown in panels A and B were then divided, and cycloheximide (CHX) was added to the indicated samples. Viability was determined by plating efficiency and is expressed as the number of CFU per milliliter as a function of time at the nonpermissive temperature. Colonies were allowed to grow on YPD plates at 25°C for 7 days before being counted. Each experiment was done at least twice, with essentially identical results. The experiment whose results are shown in panel D was done four times with identical results. Note that, in addition to the experiments described for panel D, a viability test was performed with yet another cdc33 allele, cdc33–1, using a strain with a genetic background that is different than that of CWO4. When cdc33–1 cells in SP were shifted to the nonpermissive temperature, their death curve was similar to that of the isogenic wild type (results not shown). We also note that viability tests were done also at 25°C as controls for the viability tests described for panels B and D. In these experiments, both wild-type and mutant cells remained viable for at least 1 month (results not shown).