Biological significance of the eEF2/ATP-dependent splitting of 80S ribosomes
There are physiological and genetic data consistent with the present finding that eEF2 may play an essential role in the dissociation of free 80S ribosomes in cells. Under stress conditions (3
) or during resting phase (2
), the ribosome pool is primarily preserved in the 80S form. Nutrient depletion stress dramatically increases free 80S ribosomes, and decreases the in vivo
ATP concentration (4
). In a separate experiment, the depletion of ATP elevated phosphorylation of eEF2 (36
), which inactivates the factor (37
). Similarly, the expression of a non-functional form of eEF2 (mutations H699K or V488A) led to an increase in 80S monomers (38
). All of these data point to the fact that inactivation of eEF2 with simultaneous decrease of ATP results in the accumulation of the ribosomes in the 80S form. Conversely, when the conditions are favorable for cell growth and there is enough ATP, eEF2 is activated, resulting in the rapid conversion of the accumulated 80S ribosomes to polysomes (4
The translation factors eIF3, eIF1A and eIF6 have been reported to dissociate vacant 80S ribosomes into their subunits (9
). We propose that eEF2, but not the these factors, plays the active catalytic role in the splitting of 80S ribosomes for the following six reasons. First, eIF3 is an anti-association factor that exerts its activity from the solvent side of the 40S subunit, preventing subunits joining (39
). In contrast, eEF2 translocates tRNA and mRNA through the inter-subunit space of the 80S ribosome (41
). The binding site of eEF2 is located mostly in the inter-subunit space of the 80S ribosome with domain IV contacting the broadest inter-subunit bridge B2a (42–44
). This makes eEF2 more suitable than eIF3 to get into the inter-subunits space to split the ribosome. Second, the cellular content of a core subunit of eIF3 (eIF3g) is less than the in vivo
ribosome concentration (45
). There are only 2400 molecules of eIF3i, which is also a core subunit of eIF3, per yeast cell, but 78
100 molecules of eEF2 per cell (http://www.yeastgenome.org/
). Third, the rate of the binding of eIF3 to the 40S subunit is 12-times faster than that to the 80S ribosome (26
) suggesting that the main target of eIF3 is the 40S subunit, not the 80S ribosome. Fourth, recent evidence indicates that eIF3 splits 80S ribosomes into subunits only in the presence of polyuridylic acid (46
). Fifth, the dissociation activity of eIF1A is weaker than that of eIF3 (10
). Finally, the speed of the 80S ribosome dissociation by eIF6 is incomparably slower than that by eEF2/ATP (B).
The kinetic parameters of the eEF2/ATP-dependent dissociation are consistent with the proposed physiological function. ATP stimulates binding of eEF2 to 80S ribosomes (27
). We estimated that KM
of eEF2/ATP for the 80S ribosome is 0.2
µM, which is comparable with Kd
of eEF2 for the 80S ribosome in the post-translocation state in the presence of GDP (0.4
µM) or a non-hydrolyzable analog of GTP (0.3
). The turnover number (kcat
) of eEF2/ATP for the 80S ribosome splitting is ~0.7
. This is comparable with the turnover number of the peptide bond formation in the yeast in vitro
). Due to the re-association of the split subunits into 80S ribosomes under our dissociation condition, the calculated kcat
value is much smaller than the actual turnover number of the splitting in vivo
where the formed subunits are rapidly ‘removed’ by the initiation step. In addition, our kcat
value of the ribosome/eEF2 ATPase activity (40
) is 4 times faster than kcat
of the GTPase activity of the ribosome/eEF2 complex (9.6
). Furthermore, in yeast, all ribosomes accumulate in the 80S form under conditions of glucose depletion. However, they are completely converted to polysomes within 10
min after a shift up of the culture conditions to normal glucose levels (4
). This can be explained on the basis of the known cellular content of ribosomes (45
) and eEF2, with the kcat
value we estimated for the eEF2/ATP-dependent 80S ribosome splitting.
One may argue that the spontaneous dissociation of 80S ribosomes into subunits under the optimum ionic conditions can be sufficient for the splitting, especially under the conditions where the formed subunits are efficiently ‘utilized’ for the initiation step. However, the experiment shown in B demonstrated that combining the spontaneously produced 60S subunits with eIF6, which prevents ribosome re-association, is not sufficient for rapid completion of 80S ribosome dissociation. The rate of the splitting by eIF6 is extremely slow compared with that of the splitting by eEF2/ATP.
The possibility that eEF2/ATP-dependent splitting produces inactive subunits is highly unlikely. First, eEF2 and ATP are present in vivo; it is difficult to imagine that these natural components might harm the ribosomes. Second, we have shown that the 60S subunits formed by eEF2/ATP are capable of binding eIF6 (A). Third, the split subunits can re-associate to form 80S ribosomes (A and B). However, despite these facts and the evidences cited above, the idea that the eEF2/ATP-dependent splitting of the 80S ribosomes is a part of the utilization process of the dormant ribosomes should be tested by further experiments indicating that the formed subunits can engage in the initiation and elongation phases. Until this is accomplished, our proposal will remain a hypothesis.
The dissociation of 70S ribosomes of prokaryotes is catalyzed by EF-G, GTP and RRF (8
). It is therefore reasonable to expect that eEF2, the eukaryotic homolog of EF-G, can dissociate 80S ribosomes. In a similar manner to prokaryotic IF3, eukaryotic factors (eIF3, eIF1 (11
), eIF1A or eIF6) can stabilize the ribosome subunits once they are separated by eEF2/ATP. In the experiment described in , we used eIF6 (a strong ribosome anti-association factor (13
)) as a convenient tool to keep apart the subunits formed by the eEF2/ATP-dependent reaction. Which factors play this role in vivo
is a subject of a separate study.
Since the EF-G·GTP/RRF-dependent splitting of 70S ribosomes is a part of the recycling step of the protein synthesis in bacteria, it is quite possible that the eEF2/ATP-dependent dissociation of 80S ribosomes is a part of the eukaryotic ribosome-recycling step. Our preliminary data obtained on yeast model post-termination complexes [puromycin-treated polysomes (52
)] strongly support this possibility. However, we should emphasize that in this article we do not intend to implicate the eEF2/ATP-dependent splitting of 80S ribosomes as a part of the eukaryotic recycling system until further solid evidence for this idea becomes available.
eEF2 exerts different effects on the 80S ribosome depending on the bound ligand, ATP or GTP
From the data presented in this study, it appears that eEF2/GTP exerts a different effect on ribosomes from that of eEF2/ATP. How does normal peptide elongation take place in the presence of the eEF2/ATP-dependent dissociation of 80S ribosomes? During the elongation step, the splitting reaction cannot occur because of the stabilizing effect of mRNA and peptidyl-tRNA on the ribosome. In addition, the affinity of eEF2/GTP to the pre-translocation ribosome is far greater than that of eEF2/ATP to the ribosome.
We showed that GTP inhibited the ATP-dependent splitting and the ATPase activity (). How then can eEF2/ATP mobilize 80S ribosomes in the presence of GTP? We assume that most of the free ribosomes in vivo
are in the post-translocation state. The affinity of eEF2/ATP and that of eEF2/GTP to these ribosomes are approximately the same [B and (47
)]. During shift up conditions, the concentration of ATP is ~6-fold higher than that of GTP (54
). The subunits produced by eEF2/ATP-dependent dissociation are rapidly utilized for the initiation step, driving the splitting reaction to the right by depleting the products. These considerations led us to suggest that the mobilization of the vacant 80S ribosomes by eEF2/ATP can occur in vivo
There are a number of differences in the effect of ATP and GTP on eEF2 action on the ribosome. First, fusidic acid facilitates ribosome·eEF2/GTP complex formation and keeps eEF2 bound to the ribosome (55
), thereby stimulating subunits association activity by eEF2 (unpublished data). In contrast, the effect of fusidic acid on the eEF2/ATP-dependent splitting is nominal (). Second, the binding site for ATP on eEF2 is different from that for GTP (21
). Depending on the binding site, nucleoside triphosphates exert different structural conformational changes on eEF2. In support of this hypothesis, GTP inhibited eEF2/ATP-dependent dissociation, as well as ATPase activity (). Third, GTP hydrolysis by ribosome/eEF2 is assumed to take place at the binding site of GTP on eEF2 (56
), but ribosomes may play a larger role in the hydrolysis of ATP during the dissociation by eEF2. It is known that the 80S ribosome possesses intrinsic ATPase activity (57
), which is higher than the intrinsic GTPase activity. This ATPase is presumably associated with the 5S RNP complex and is stimulated by eEF2 (59
). The central protuberance, which involves the 5S RNP complex and forms B1a and B1b/c inter-subunit bridges (43
), undergoes substantial conformational changes upon binding of eEF2/sordarin (44
). These data suggest that eEF2/ATP-dependent splitting of the 80S ribosome might be triggered by the conformational change of the central protuberance of the 60S subunit.
It is known that ATP is required for the initiation (60
) and elongation (62–64
) steps of polypeptide synthesis. In this study, we show that ATP is also consumed for splitting of 80S ribosomes into subunits, which may be important for utilization of 80S ribosomes for the initiation step. Thus, in contrast to prokaryotic protein synthesis, ATP plays critical roles in eukaryotic translation.