The scientific literature contains a number of observations that can be used for the purpose of cross-referencing and validating the results obtained in this study. In particular, eIF4A has previously been described as the most abundant initiation factor (Neff and Sachs, 1999), and a roughly equimolar occurrence of eIF4E and p20 with the ribosomes was observed, but a lower abundance of eIF4G (Altmann et al., 1997). Correlation of our protein values with published mRNA copy numbers for initiation factors generally supports the relative ratios between eIFs that we found. The factor with the highest protein copy number also has the highest mRNA abundance, and the converse relationship exists for the two least abundant eIFs (Fig. 3). The ratios for the other factors do not correlate as well, but are within published ranges for mRNA:protein ratios (Futcher et al., 1999).
Although yeast cell volume can change considerably, for example as a function of carbon source (Tyson et al., 1979), analyses under conditions very similar to ours have consistently found volumes of 35–40 µm3 (Tyson et al., 1979; Russo et al., 2000; 2001). Of this total volume, 25% has been estimated to be solid material (Dombek and Ingram, 1986). Accordingly, the intracellular volume available to be occupied by macromolecules corresponds to 25–30 µm3 per cell, assuming an initial value of 36 ± 3 µm3 (Tyson et al., 1979). With the content of extractable protein estimated by us to be 5 mg per 109cells, this would result in total intracellular protein concentrations of 170–200 mg ml−1, which compares to published values of 200–300 mg ml−1 cytoplasmic protein for E. coli (Zimmerman and Trach, 1991). These calculations neglect the fact that yeast as a eukaryote is organized into compartments (including vacuoles), which may contain significantly different densities of macromolecules. They are intended to demonstrate that we are unlikely to have made any gross errors in our calculations of cell numbers and protein concentrations.
The results presented here were obtained through application of a single method, and may therefore suffer from inherent inaccuracies. Also, the concentrations of specific proteins are likely to be at least as variable as is cell size between different populations. Taking these considerations into account, the minimal interpretation of the data presented in Fig. 2 is to divide yeast translation initiation factors into three groups: those that are significantly more abundant than ribosomes (eIF4A), those that are less abundant (eIF1A, eIF2Bα and eIF4G), and those that are roughly comparable to ribosome levels (i.e. within a range covering 0.5–2.0 × the level of ribosomes: eIF1, 2α, 2β, 3c, 4B, 4E and p20). Even when interpreting our findings in such a broad way, they have interesting implications for interactions that occur between eIFs.
Leaving aside the factors involved in cap-complex formation (the group 4 factors and p20), the groups identified above by means of abundance relate in a fully explicable way to the structural and non-structural roles of the respective factors during formation of the 43S complex. All factors occurring at levels comparable to the ribosomes have been previously identified as integral parts of the 43S complex (Asano et al., 2000). In contrast, eIF2B is well characterized as an enzyme that regenerates the eIF2:GDP complex produced after each initiation event (reviewed in Webb and Proud, 1997). The notion that this factor acts as an enzyme is consistent with the requirement for a lower abundance compared to its proposed substrate. A similar situation may be true for eIF1A, which was shown to catalyze the eIF2-dependent transfer to the small ribosomal subunit (Chaudhuri et al., 1997). Alternatively, this factor may constitute a limiting structural component of 43S complexes.
Observations made after glucose withdrawal from logarithmically growing cells show that yeast can progress from the absence of translation to normal polysomal profiles within a very short time (Ashe et al., 2000). The comparable ratios between ribosomal subunits and the other 43S complex components are consistent with the ability to rapidly commit the entire 40S pool to translation. The observed equimolarity would allow most or all of the 40S subunits to simultaneously exist as 43S complexes, omitting the need to wait for a first round of ribosomes to finish initiation, eIF2 to be regenerated, and a second round of 43S complexes to be formed.
In contrast to the proteins discussed above, the observed ratios between the eIF4 factors are less straightforward to explain. In the case of eIF4A, the very high abundance is consistent with observations that this protein has relatively weak helicase activity compared to other members of the DEAD-box family (de la Cruz et al., 1999), and that it was proposed to function in translation in a manner that involves cycles of transient binding to a more stably bound eIF4E:eIF4G complex (Yoder-Hill et al., 1993; Pause et al., 1994). Thus, the large amount of intracellular eIF4A would be required to enable delivery of a sufficient level of helicase-activity to mRNA 5′ ends, a notion that is supported by the observation that addition of eIF4A to yeast cell-free extracts increases translational activity despite the high initial levels of this protein (Neff and Sachs, 1999). Interestingly, eIF4A helicase-activity is stimulated by eIF4B in vitro (Rozen et al., 1990); but the eIF4A:eIF4B ratios reported here indicate that this stimulation can only apply to a subset of eIF4A molecules at any given time. It is known that eIF4A can function in translation independently of eIF4B, as strains unable to produce the latter protein are viable (Coppolecchia et al., 1993). Taken together, these findings suggest that eIF4A-related RNA helicase activity may be delivered in two different modi in vivo, partially by eIF4A alone, and partially by eIF4B-stimulated eIF4A.
Assuming again a total intracellular volume available to proteins of 25–30 µm3, the concentration of ribosomes in yeast would be as high as 11–13 µM; that of eIF4A would be 50–60 µM and the concentration of eIF4G or eIF2B would still be in the µM range (Fig. 2B). Binding partners interacting with equilibrium constants in the low to mid nM range (kDa = 10−9–10−7), such as eIF4E:eIF4G (Ptushkina et al., 1998), or eIF3:eIF1 (von der Haar et al., 2002), would therefore be very close to existing fully in the bound state at any given time provided that these interactions are not regulated by other proteins.
More interesting still is the consequence of such high concentrations for weaker interactions. Transient interactions, and the ability to change readily from a bound to an unbound state, are an inherent requirement of a model of translation initiation in which one ribosome after another is recruited to an mRNA by the action of initiation factors. Affinities in the range of 1–10 µM best fit this specification for the assumed factor concentrations, whereas stronger affinities would make it less likely for a protein to spend significant time in the unbound state. Conversely, weaker interactions would reduce the likelihood of efficient recognition of the binding partner. However, it is important to note that according to thermodynamic theory such calculations have to be based on activities rather than concentrations. An important next step in assessing the impact specific translation initiation factor levels have on translational regulation will therefore be the experimental determination of coefficients that relate concentration to activity in the densely crowded intracellular environment.
In conclusion, the eIF quantification performed in this study has generated information of key significance to our understanding of translation in a eukaryotic model organism. The numbers obtained from this work con-stitute an important guide for the next major step in research on the yeast translation system, which will be the full reconstitution and quantitative analysis of its components.
Intracellular eIF4E concentrations and translation
As has been noted above, the levels of eIF4E, eIF4G and p20 reported here cannot easily be related to our current models of translation initiation. According to the commonly accepted model, supported by an extensive body of evidence, eIF4E functions in a 1 : 1 complex with eIF4G to build a molecular bridge between the mRNA 5′ cap and the 43S complex. If this is the case, why does yeast maintain an almost 10-fold excess of eIF4E over eIF4G? Theoretically, an excess of eIF4E might be important for translation initiation, e.g. in order to enforce high rates of complex formation; alternatively, eIF4E may be required independently of eIF4G in other areas of mRNA metabolism (for a review of roles of eIF4E outside translation see Strudwick and Borden, 2002). Our experiments investigating the effects of limitation of intracellular eIF4E levels by promoter substitution very clearly show that the bulk of ongoing translation under logarithmic growth conditions does not require most of the available eIF4E. This finding is consistent with previous studies showing that cap-binding activity of eIF4E can be considerably weakened by mutagenesis without severely affecting cell viability (Vasilescu et al., 1996), which had already indicated that eIF4E does not exert particularly strong rate limitations on translation in yeast cells. Moreover Lang et al. (1994) showed that changes in the in vivo abundance of eIF4E over a broad range have little effect on cell viability. Here, we quantify for the first time the extent to which a reduction in eIF4E levels can be tolerated, in that more than two thirds of eIF4E can be lost without affecting either growth rate or translational activity.
In view of the assumed analogy between the mammalian 4E-BPs and yeast p20, it was surprising to find that p20 is associated with eIF4E during logarithmic growth. The suggestion that p20 has the same function of translational repressor as the 4E-BPs have in mammals was based mainly on the biochemical similarities between the two proteins: both are small phosphoproteins, they can bind eIF4E via motifs that are very similar to each other and motifs present in eIF4G, and when doing so, they displace eIF4G, thereby rendering eIF4E inactive for translation (Altmann et al., 1989; 1997; Zanchin and McCarthy, 1995; Ptushkina et al., 1998). However, in contrast to the 4E-BPs, it has never been shown for p20 that association with eIF4E depends on the translational status of the cell, or on the phosphorylation status of p20.
The association between p20 and eIF4E under conditions of high translational activity suggests that its main function may not be as translational inhibitor. Analysis of CAF20 deletion strains, and of strains containing reduced eIF4E levels, shows that the presence of eIF4E:p20 complexes is not required for the control of translation under standard growth conditions, nor for any other aspect of cellular function investigated so far. Because we could not detect association of p20-bound eIF4E with poly(A) mRNAs in wild-type yeast during logarithmic growth, we conclude that logarithmically growing yeast maintains eIF4E in two functional pools: one, bound to eIF4G, is necessary for translation of all capped mRNAs. The other, bound to p20, is not required for translation at least of the majority of mRNAs. Although the eIF4E normally bound in this pool is not essential for bulk translation and normal growth rates, it or part of it is essential for the maintenance of normal cellular morphology: this may be because it is necessary for translation of particular mRNAs with higher requirements on eIF4E activity, such as the CLN3 transcript (Danaie et al., 1999), or because it has roles outside of translation.
Although the pool of non-eIF4G-bound eIF4E is bound to p20 in wild-type yeast cells, it can exist independently of the latter protein if this is absent from the cells. Therefore, in contrast to the eIF4E contained in the pool of eIF4E:p20 complexes, p20 itself appears not to be involved in cellular fitness under any of the conditions tested. So far, the only clear phenotype observed when levels of p20 and eIF4E are co-ordinately increased is a modulation of half-lives of mRNAs in vivo, presumably stemming from competition with the mRNA degradation machinery (Velasco et al., 2002). It would therefore be interesting to test whether p20 has a function under conditions where RNAs have to be protected from the degradation machinery in the absence of translation. An example for such a situation is sporulation, during which the CAF20 gene was shown to be transcriptionally regulated (Chu et al., 1998).
A last point of discussion is whether the situation described here for the cap-binding complex of budding yeast is specific to this organism or applies to eukaryotes in general. p20 appears to be significantly different from the 4E-BPs in that it is associated with eIF4E even under conditions of the highest translational activity, in complexes that are not bound to polyadenylated mRNAs. A role for this protein, whatever it may eventually turn out to be, is therefore possibly specific to yeast.
In terms of the excess of eIF4E over eIF4G, we cannot yet determine whether this situation is particular to yeast or occurs also in other cells. To our knowledge, the only other study that has investigated intracellular levels of eIF4E and eIF4G concerned HeLa cells, where the situation appears reversed compared to yeast with a twofold excess of eIF4G over eIF4E (Duncan et al., 1987). However, changes in the levels of both eIF4E and eIF4G have been reported for many cancers (Dua et al., 2001; Watkins and Norbury, 2002), so that the ratios for these factor may be different in non-transformed cell lines. Indeed, it has previously been questioned whether a general description of eIF4E as the rate limiting factor in translation is true even for mammalian cells (Rau et al., 1996, also discussed in McCarthy, 1998).