Eukaryotic cells utilize multiple eIF2B targeting pathways (Proud, 2005
). Previously, we have characterized S180 and P180 allelic variants in the yeast eIF2Bγ (GCD1
gene), which confer sensitivity and resistance to fusel alcohols (e.g., 1-butanol), respectively (Ashe et al., 2001
). Here, we find that both strains are translationally inhibited by butanol, yet the butanol concentration required to achieve inhibition differs dramatically. Such differential sensitivity is not observed for ethanol (data not shown) but is seen for other alcohols, including well-characterized products of branched-chain amino acid catabolism (isoamyl alcohol and isobutyl alcohol; Hazelwood et al., 2008
). Therefore, it seems likely that there is some general mechanism where alcohols of a certain carbon chain length elicit effects on translation.
In our initial study, a panel of eIF2B mutants exhibited butanol-dependent phenotypes (Ashe et al., 2001
). Furthermore, butanol-sensitive phenotypes have since been observed for specific eIF2B VWM mutants in yeast (Richardson et al., 2004
). In this current study, we have characterized an eIF2Bε mutant, gcd6-1
(Bushman et al., 1993a
). As a Gcd−
mutant, this strain is slow growing and has reduced basal translation rates. Somewhat surprisingly, we found that this mutant is butanol resistant, and we identified C383W as the mutated residue. This mutation impacts on one of the eIF2Bε hexapeptide repeats that constitute the left-handed parallel LbH domain (Aravind and Koonin, 2000
). This domain is also present in eIF2Bγ, so we generated the analogous eIF2Bγ-C483W mutant strain, which exhibited extreme sensitivity to fusel alcohols. Therefore, although the LbH domains of both eIF2Bε and γ are important for the fusel alcohol response, it is unlikely that they function similarly. The LbH domain is found in a variety of acyltransferases and is generally involved in the formation of trimeric complexes (Johnson et al., 2005
), although other conformations are possible (Gorman and Shapiro, 2004
). However, the relevance and functions of this domain and its structure in the context of the eIF2B heteropentamer are currently unknown.
Two butanol-resistant eIF2Bα mutations (GCN3-R148K
) were identified using an oligonucleotide array strategy. Mutation at T41 has previously been described as a Gcn−
mutant (Pavitt et al., 1997
), whereas mutation of R148 has not been characterized before. Both residues are well conserved, and both R148 and T41 are present in human eIF2B1 (data not shown). Both mutations phenocopy the gcn3
Δ strain after amino acid starvation, because they fail to inhibit translation initiation. In stark contrast, the butanol response is unaltered in a gcn3
Δ mutant, whereas the two GCN3
mutants are butanol resistant. These genetic intricacies probably reflect the complicated subunit interactions and regulation of this factor. Overall, eIF2Bα, which is required for the inhibition of translation initiation as a result of eIF2α phosphorylation, is also a key player in the fusel alcohol response.
In contrast to other translational inhibitory mechanisms, eIF2α becomes dephosphorylated in response to fusel alcohols. This was unexpected given that the kinetics and scale of translational inhibition are virtually identical after either fusel alcohol addition or amino acid starvation (which induces eIF2α phosphorylation; Smirnova et al., 2005
). Two phosphatase deletion mutants, ptc2
Δ and sit4
Δ, exhibited unusual eIF2α phosphorylation profiles. The ptc2
Δ mutant exhibited high basal phosphorylated eIF2α levels that were not further elevated upon amino acid starvation. Ptc2p is a type 2C phosphatase that dephosphorylates Hog1p and Ire1p, influencing the osmotic stress and unfolded protein responses, respectively (Welihinda et al., 1998
; Young et al., 2002
). Ptc2p also influences the yeast cell cycle via Cdc28p (Cheng et al., 1999
) and the DNA checkpoint pathway (Guillemain et al., 2007
). The fact that a ptc2
Δ mutant has constitutively high phospho-eIF2 levels that are unresponsive to amino acid starvation suggests that this strain harbors nonregulatable Gcn2p kinase. Therefore, it is possible that in a wild-type strain, the Ptc2p phosphatase acts on Gcn2p to inhibit or dampen the basal kinase activity. However, Ptc2p is clearly not required for the fusel alcohol–induced dephosphorylation of eIF2α.
In the case of the type 2A–related phosphatase Sit4p, a deletion mutant exhibits virtually no eIF2α dephosphorylation after exposure of yeast cells to fusel alcohols. Therefore, it seems likely that Sit4p is activated after fusel alcohol treatment and either directly or indirectly leads to a decrease in phosphorylated eIF2. However, in the sit4
Δ mutant, translation initiation is still inhibited after fusel alcohol treatment; therefore, neither the activation of Sit4p nor the dephosphorylation of eIF2 is involved in the effects of fusel alcohols on translation initiation. However, in a physiological sense the dephosphorylation of eIF2α observed after fusel alcohol treatment may prevent the inappropriate activation of an amino acid starvation cellular response. This might be especially important given that fusel alcohol stress can be viewed as metabolically opposed to amino acid starvation; i.e., fusel alcohols signal the catabolism of amino acids as cells desperately attempt to find nitrogen sources, whereas amino acid starvation signals the up-regulation of anabolic pathways involved in channelling nitrogen reserves toward the biosynthesis of amino acids. The activation of Sit4p in response to fusel alcohols is also compatible with other responses that are activated by this stress, such as the pseudophyphal response (Lorenz et al., 2000
). Indeed, a sit4
Δ mutant is incapable of undergoing pseudohyphal growth after nitrogen starvation (Cutler et al., 2001
Intriguingly, volatile anesthetics, like fusel alcohols, lead to the dephosphorylation of eIF2α and GCN3
mutants are resistant to these compounds (Palmer et al., 2005
). This suggests that fusel alcohols and volatile anesthetics impact on translation initiation in a remarkably similar manner. However, subtle differences are apparent. For instance, gcn3
Δ strains are more resistant to volatile anesthetics, whereas we show that deletion of GCN3
produces little difference in the response to fusel alcohols. Similarly, a gcn2
Δ mutant is resistant to volatile anesthetics, whereas GCN2
deletion does not affect the fusel alcohol response. In response to volatile anesthetics, the impact of the GCN2
deletion was dependent on the strains' auxotrophic status. We observe no correlation between auxotrophy/prototrophy and the effects of fusel alcohols. Therefore, these two reasonably diverse sets of chemicals target protein synthesis via very similar but not entirely identical mechanisms (Ashe et al., 2001
; Palmer et al., 2005
Recently we have found that eIF2B and eIF2 reside in a large cytoplasmic body that we have termed the “2B body” and that this body represents a site where guanine nucleotide exchange of eIF2-GDP to eIF2-GTP can occur (Campbell et al., 2005
). Fusel alcohols alter a variety of properties with regard to this 2B body. For instance, they impact on the rate of eIF2 transit through the body. This rate has previously been shown to vary in a manner that equates to the level of guanine nucleotide exchange activity after stress or mutation (Campbell et al., 2005
). However, fusel alcohols cause much less pronounced changes than other stresses that inhibit eIF2B and translation initiation. The explanation for this is not clear, but we favor the hypothesis that nonproductive eIF2 transit may occur where the rate of exchange is regulated by mechanisms other than increased eIF2α phosphorylation. Thus the rate of eIF2 transit through the 2B body would be an overestimate of the guanine nucleotide exchange activity. Fusel alcohols also increase the length of time that the body remains in a tethered state, thus reducing the movement of the body around the cell. This effect is more exacerbated in a BUTS
strain relative to a BUTR
strain. The GCN3
mutants that suppress a BUTS
mutation do not possess eIF2B bodies and therefore in these strains eIF2B is unlikely to be present in a tethered state. Interestingly, these data have implications for the function of the eIF2B body. The GCN3
mutant strains that lack the eIF2B body show no decrease in growth or protein synthetic rates, suggesting that the eIF2B body is not required for maximal protein synthesis. However, these strains are resistant to the effects of both amino acid starvation and fusel alcohols on translation initiation. Therefore, it is possible that the concentration of eIF2B into a large body permits a greater degree of regulation and, in the absence of this body, regulatory pathways are compromised.
In terms of the mechanism by which fusel alcohols inhibit eIF2B to generate reduced ternary complex, it is not clear whether the regulation is direct or indirect. Direct regulation of enzymes by alcohols has been described previously. For example, alcohol-binding sites exist in the cysteine-rich domains of various isoforms of protein kinase C, and depending on the precise nature of the bound molecule, they have been shown to both stimulate and inhibit kinase activity (Stubbs and Slater, 1999
). Intriguingly, these sites overlap with the anesthetic-binding sites (Das et al., 2006
). Therefore, it is possible that the eIF2B guanine nucleotide exchange factor represents a point of regulation for alcohols and volatile anesthetics as a result of such a direct binding reaction. Alternatively, the alcohols could interact and impact on a component of a signaling pathway, and one output of this pathway would be the posttranslational modification of eIF2B. In both of these cases, either the direct binding of alcohols or the posttranslational modification of eIF2B would alter the guanine nucleotide exchange activity of this complex, reducing translation initiation. Another consequence of this inhibition of the guanine nucleotide exchange activity would be increased tethering of the eIF2B body. If true, this would implicate the exchange of guanine nucleotides as important in the release of the eIF2B body from the tethered state.
Alternatively, it is possible that fusel alcohols either directly or indirectly impact on the process by which the eIF2B body becomes untethered. In this model, the increased tethering would lead to reduced guanine nucleotide exchange activity even though the intrinsic activity of eIF2B would remain unaltered. By preventing the movement of the eIF2B, fusel alcohols could locally generate increased eIF2-GTP and decreased eIF2-GDP levels. The decrease in substrate and increases in product could potentially lead to the inhibition observed here.
Overall, this study illustrates that eIF2B is targeted by fusel alcohols to generate reduced ternary complex levels and translation initiation rates in a manner that is similar to the effects of volatile anesthetics. This work also emphasizes the 2B body as an element of the cell biology of protein synthesis that might be fine-tuned as a consequence of cellular stress.