InteractiveFly: GeneBrief

eukaryotic translation initiation factor 4E homologous protein: Biological Overview | References

BIOLOGICAL OVERVIEW

In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally-derived mRNAs, initiates pattern formation. For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis. Inhibition of hb mRNA translation requires an mRNP complex (the NRE-complex) that consists of Nanos (Nos), Pumilio (Pum) and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3' UTR of hb mRNA. The identity of the mRNA 5' effector protein that is responsible for this translational inhibition remained elusive. This study shows that d4EHP, a cap-binding protein which represses caudal (cad) mRNA translation (Cho, 2005), also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5' cap structure (m⁷GpppN, where N is any nucleotide) and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo (Cho, 2006).

Transcription is globally repressed in the rapidly-dividing nuclei of early Drosophila embryos, and therefore gene expression is largely regulated by translational control of maternally-provided mRNAs. Translation is often regulated at initiation, which occurs in multiple steps starting with the recruitment of the 40S ribosomal subunit to the 5' end of an mRNA and resulting in the correct positioning of the 80S ribosome at the initiation codon. Recognition of the cap structure by eIF4F (composed of three subunits: eIF4E, eIF4A and eIF4G) is an integral part of this process. Moreover, eIF4G interacts both with eIF4E and the poly(A)-binding protein (PABP), thus circularizing the mRNA, which in turn is believed to promote re-initiation. Consistent with their importance, eIF4E and PABP have emerged as major targets of translational regulatory mechanisms mediated by such modulator proteins as 4E-BPs and Paip2 (Cho, 2006).

Embryonic development in many metazoans requires the activity of various maternal determinants called morphogens, whose spatial and temporal expression is tightly regulated. In Drosophila, local morphogen concentrations are important for the establishment of polarity and subsequent organization of both the antero-posterior and dorso-ventral axes of the embryo. A key morphogen for antero-posterior patterning is the transcription factor Hunchback (Hb); when maternal Hb is allowed to accumulate inappropriately, posterior segmentation is blocked. Two modes of translational control have been proposed for the establishment of the maternal Hb gradient: translational silencing via deadenylation and inhibition at the initiation step in a cap-dependent manner (Chagnovich, 2001; Cho, 2006 and references therein).

d4EHP, an eIF4E-like cap-binding protein that does not interact with deIF4G and d4E-BP, inhibits the translation of cad mRNA by interacting simultaneously with the cap and Bicoid (Bcd) (Cho, 2005). While many embryos (~41%) produced by females homozygous for the d4EHP^CP53 mutation showed anterior patterning defects consistent with mislocalized Cad, some (~7%) also exhibited patterning defects such as missing abdominal segments that cannot be readily explained by ectopic Cad expression. Since inhibition of hb mRNA translation has been linked in one study to the cap structure (Chagnovich, 2001) and since these additional phenotypes could be consistent with inappropriate regulation of Hb, this study investigated the role of d4EHP in Hb expression. Embryos (0-2h) from females homozygous for the d4EHP^CP53 mutation (Cho, 2005) were collected and immunostained using anti-Hb antibody. DNA was stained with DAPI to highlight the nuclei). For simplicity, embryos will subsequently be referred to by their maternal genotype. To evaluate the extent of the Hb gradient its signal intensity was measured at 38-50 locations along the anterior-posterior axes of 6-16 embryos of each genotype. The values were corrected for overall signal intensity and then normalized the data for embryo length (EL, anterior pole = 0%, posterior pole = 100%). The normalized values were plotted and average intensity values were calculated to obtain an average trend. It was observed that in Ore^R embryos, Hb signal intensity drops steeply in the middle of the embryo and reaches 50% maximum intensity at 48% EL. In d4EHP^CP53 embryos the Hb expression domain extended substantially further toward the posterior and signal intensity remained at approximately 50% of the maximum throughout the region between 50-75% EL. Normal Hb distribution was restored to d4EHP^CP53 mutant embryos by transgene-derived expression of wild-type d4EHP (d4EHP^wt, but not by expression of a mutant form of d4EHP (d4EHP^W114A), which is unable to bind the cap structure. Expression of another form of d4EHP (d4EHP^W85F) which cannot bind Bcd, fully rescued the defective Hb gradient. The expression levels of the wild-type and mutant d4EHP transgenes are essentially equal. Distributions of Nos, Pum, and Brat were unaffected in d4EHP^CP53 mutant embryos. Taken together, these data demonstrate that d4EHP plays a key role in establishing the posterior boundary of Hb expression in a manner that requires its cap-binding activity but not an association with Bcd (Cho, 2006).

It was reasoned that Brat might be a candidate partner protein for d4EHP since both are relevant for hb regulation. Thus whether d4EHP and Brat physically interact was investigated in vivo. Extracts prepared from 0-2h Oregon-R (Ore^R) embryos were treated with RNase and used to examine the interaction between Brat and d4EHP. Western blotting analysis using antibodies against d4EHP and Brat demonstrates that, while anti-d4EHP co-immunoprecipitated endogenous Brat, pre-immune serum did not. To further demonstrate the specificity of this interaction, HA-tagged deIF4EI and the RNA-binding protein La (negative controls) were transfected in HEK293 cells along with FLAG-tagged full-length Brat. While anti-FLAG antibody immunoprecipitated wild-type HA-d4EHP together with FLAG-Brat, deIF4EI and La failed to co-immunoprecipitate. Similarly, other RNA-binding proteins such as hnRNP U and HuR, and a d4EHP mutant (W173A), in which a tryptophan residue that is part of the hydrophobic core and thus affects protein folding is replaced, also failed to interact with Brat, demonstrating that Brat interacts specifically with d4EHP. Since a cell transfection system was used to assay for the d4EHP:Brat interaction, it is possible that other bridging proteins are required for the d4EHP-Brat association (Cho, 2006).

To identify the Brat-interacting domain of d4EHP, a number of individual residues located on its convex dorsal surface were mutated, and co-immunoprecipitation was tested with Brat. From this work no point mutant of d4EHP was identified that abrogated the interaction. As an alternative approach, chimeric proteins were created in which different domains of d4EHP were replaced with their counterparts from deIF4EI, taking advantage of the knowledge that, unlike d4EHP, deIF4EI does not interact with Brat. Mhree mutant forms of d4EHP were produced, with each one of its three dorsal α-helices replaced with that of deIF4EI. It was found that, while helix 1 and 2 mutants failed to disrupt binding to Brat, replacement of d4EHP helix 3 (residues 179 to 194) significantly reduced the interaction with Brat. Consistent with these observations, α-helix 3 is the most divergent between d4EHP and deIF4EI. The overall structure of d4EHP is not affected by the replacement of helix 3 with its deIF4EI counterpart, since the chimeric protein still binds to the cap. Thus, these data demonstrate that Brat interacts with d4EHP on its convex dorsal surface and that this interaction is mediated by the third α-helix of d4EHP (Cho, 2006).

A C-terminal domain of Brat termed the NHL domain is both necessary and sufficient to inhibit hb mRNA translation (Sonoda, 2001). The NHL domain contains two large surfaces (defined as top and bottom), that can support protein-protein interactions (Edwards, 2003). While the top surface of the NHL domain binds to Pum and Nos, the bottom surface does not interact with any known protein. Although the Brat NHL domain contains an amino acid sequence that conforms to the YxxxxxxLΦ d4EHP-binding motif (Cho, 2005), the d4EHP:Brat interaction does not require this motif, since a Brat deletion mutant that lacks it can still interact with both d4EHP and the d4EHP W85F mutant. This sequence is most probably masked from interaction with d4EHP because it is located in the hydrophobic core of the NHL domain. To determine whether the d4EHP:Brat interaction requires the NHL domain, a Brat mutant that lacks the domain (Brat ΔNHL) was engineered and used in a co-immunoprecipitation experiment. While wild-type Brat was readily co-immunoprecipitated with d4EHP, the Brat ΔNHL mutant was not. Thus, it is concluded that the NHL domain is the site of d4EHP interaction. To further characterize this interaction, point mutations were designed to replace residues on the two surfaces of the NHL domain, and the mutant proteins were tested for their ability to interact with d4EHP. Mutation of a top surface residue that affects Brat interaction with Pum (G774A) did not affect the d4EHP:Brat interaction. However, when residues on the bottom surface were mutated, the d4EHP:Brat interaction was either significantly reduced (G860D and KE809/810AA), or abrogated (R837D and K882E). Importantly, the Brat NHL R837D mutant can assemble into an NRE-complex, demonstrating that this mutation specifically affects the d4EHP interaction and not the interactions with Pum and Nos (Cho, 2006).

Brat inhibits hb mRNA translation by interacting with the NRE-complex (Sonoda, 2001). Since d4EHP interacts physically with Brat, it was asked whether d4EHP can be co-purified with the NRE complex in vitro. Incubation of recombinant components of the NRE-complex (Brat, Pum, Nos and NRE) together with HA-tagged d4EHP resulted in the retention of d4EHP on glutathione-Sepharose beads through the GST-Pum RNAB fusion protein. The association of Brat with d4EHP was dependent on the ability of d4EHP to bind to Brat, since addition of Pum/Nos/NRE alone or in combination with the Brat R837D mutant failed to capture it. Thus, by interacting with Brat, d4EHP can associate with the NRE complex (Cho, 2006).

To investigate the biological significance of the d4EHP:Brat interaction, the effects of Brat mutants, which are defective for d4EHP binding, were examined in Drosophila embryos. brat^fs1 mutant embryos exhibit a significant expansion of the Hb expression domain towards the posterior and display severe abdominal segmentation defects. When a brat^WT transgene is expressed in the brat^fs1 mutant background, normal Hb distribution and a wild-type segmentation pattern is restored). To investigate whether interaction with d4EHP is essential for the function of Brat in embryonic patterning, transgenes were introduced encoding mutant forms of Brat that affect the d4EHP:Brat interaction (brat^R837D and brat^K882E) into the brat^fs1 mutant background. Despite being expressed at levels similar to the brat^WT transgene, these mutant forms fail to fully rescue the normal Hb gradient and, importantly, do not fully rescue the brat^fs1 mutant phenotype. Taken together, these data strongly argue that the d4EHP:Brat interaction contributes significantly to hb regulation (Cho, 2006).

Through its interaction with Brat, d4EHP defines and sharpens the posterior boundary of Hb expression. Based on the hypomorphic d4EHP^CP53 phenotype, its activity appears most relevant to hb regulation in the region of the embryo from 50-75% EL, although it is possible that a null d4EHP allele would have more drastic effects. The d4EHP:Brat interaction is mediated via residues on the bottom surface of the Brat NHL domain. Thus, as in the established for cad (Cho, 2006), a simultaneous interaction of d4EHP with the cap and Brat results in mRNA circularization and renders hb translationally inactive. Since the interaction between Brat and d4EHP does not involve the previously described 4EHP-binding motif (YxxxxxxLΦ), it is possible that d4EHP interacts with Brat through a bridging protein (Cho, 2006).

The data support a model for the requirement for the 5' cap structure in regulation of endogenous hb mRNA. This is consistent with an earlier study that assessed translation of NRE-containing mRNAs after injection into Drosophila embryos and concluded that the cap structure is functionally significant (Chagnovich, 2001). In contrast, another study reported that Nos and Pum repressed the expression of an engineered transgene containing an internal ribosome entry site (IRES) and a hairpin loop designed to block cap-dependent translation (Wharton, 1998). These results were used to conclude that hb translational repression is cap-independent. However, the phenotypic assay used in that study was indirect and the observed results could also be caused by RNA destabilization. Furthermore, Nos-dependent deadenylation was also shown to be important in establishing the Hb gradient (Wreden, 1997). It is difficult to reconcile all these data without concluding that multiple distinct post-transcriptional mechanisms regulate Hb expression, including two that require Nos. The novel d4EHP-dependent mechanism defined in this study appears important for repressing hb in more central regions of the embryo, while cap-independent regulation involving deadenylation of hb mRNA may predominate in more posterior regions of the embryo. It is noted that mutant forms of Brat that are abrogated for d4EHP interaction retain substantial (but not complete) activity in repressing hb, suggesting some redundancy between these two mechanisms. Analogous overlapping translational control mechanisms have recently been reported for Bruno, which represses Oskar (Osk) expression (Chekulaeva, 2006) both through cap-dependent translational regulation and through packaging osk mRNA into translationally silent RNP complexes (Cho, 2006).

Identification of a common inhibitory mechanism which regulates cad and hb mRNA translation simplifies the understanding of how the anterior-posterior axis is organized during early Drosophila embryogenesis. By regulating two classical maternal morphogenetic gradients, d4EHP plays a critical role in early Drosophila embryonic development. It is noteworthy that d4EHP is recruited to these mRNAs through different RNA binding proteins that presumably recognize different sequence elements. In the case of cad, d4EHP becomes associated by binding directly to Bcd, which in turn recognizes a defined 3’UTR element, the BBR. In the case of hb, Bcd binding is not involved in d4EHP recruitment and no element similar to the BBR is present. It remains uncertain whether the interaction between d4EHP and Brat is direct or indirect; since d4EHP and Brat are both uniformly distributed in early embryos, a non-uniformly distributed bridging protein mediating this interaction may be the basis of the spatially-restricted requirement for d4EHP in hb repression. Since d4EHP and some of its interacting partners are evolutionarily conserved in higher eukaryotes and because cap-dependent translation regulation plays such an important role in eukaryotic gene expression, it is predicted that 4EHP-dependent translational inhibitory mechanisms are widespread throughout the animal kingdom (Cho, 2006).

A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP

Translational control is a key genetic regulatory mechanism implicated in regulation of cell and organismal growth and early embryonic development. Initiation at the mRNA 5' cap structure recognition step is frequently targeted by translational control mechanisms. In the Drosophila embryo, cap-dependent translation of the uniformly distributed caudal (cad) mRNA is inhibited in the anterior by Bicoid (Bcd) to create an asymmetric distribution of Cad protein. d4EHP, an eIF4E-related cap binding protein, specifically interacts with Bcd to suppress cad translation. Translational inhibition depends on the Bcd binding region (BBR) present in the cad 3' untranslated region. Thus, simultaneous interactions of d4EHP with the cap structure and of Bcd with BBR renders cad mRNA translationally inactive. This example of cap-dependent translational control that is not mediated by canonical eIF4E defines a new paradigm for translational inhibition involving tethering of the mRNA 5' and 3' ends (Cho, 2005).

This study describes a new mode of mRNA-specific translational inhibition, which acts by tethering the mRNA 5' and 3' end via d4EHP, an eIF4E-related protein, and Bcd. d4EHP binds to the cad mRNA 5' cap structure, while Bcd binds to BBR in its 3' UTR. The interaction between d4EHP and Bcd is mediated through a sequence motif in Bcd that resembles, but is distinct from, the consensus eIF4E binding domain present in classical eIF4E binding proteins such as 4E-BPs and eIF4G. Inhibition of cad mRNA translation by the d4EHP:Bcd complex demonstrates for the first time the involvement of a cellular cap binding protein other than eIF4E in cap-dependent translational control. Furthermore, it provides a new molecular mechanism governing the formation of morphogenetic gradients during early Drosophila embryo development (Cho, 2005).

It was previously reported that Bcd inhibits anterior Cad synthesis through a direct interaction with eIF4E (Niessing, 2002). This conclusion was based largely on an in vitro demonstration that Bcd could be recovered from Drosophila extracts using a cap-affinity resin, which was prebound to an excess amount of recombinant eIF4E. However, under these conditions, only a small fraction of Bcd was recovered from the extracts. It is therefore a distinct possibility that Bcd actually bound to the cap-affinity resin through endogenous d4EHP that was also present in the extracts. This possibility is consistent with both the previous data and the present study. Further supporting this conclusion, endogenous deIF4E and Bcd were not shown to interact in the previous study. The data also indicate that the L73R mutation alone is sufficient to explain the previously reported bcd^Y68A/L73R double mutant phenotype (Cho, 2005).

The role of 4E-BPs in regulating cap-dependent translation is well documented. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E and are therefore general inhibitors of cap-dependent translation, although the degree of inhibition varies among different mRNAs. Cup and Maskin are eIF4E binding proteins that regulate translation during oogenesis and embryonic development. They inhibit the translation of specific mRNAs by a simultaneous interaction with eIF4E at the mRNA 5' end and proteins bound to sequence elements in the 3' UTR. Thus, Cup and Maskin have to compete with eIF4G for binding to eIF4E. While the exact binding affinities of these proteins for eIF4E have not been determined, it is known that Maskin interacts rather weakly with eIF4E (Cho, 2005).

In contrast to 4E-BP, Cup, and Maskin, Bcd does not need to compete with eIF4G to interact with d4EHP. Rather, it is d4EHP that competes with eIF4E for cap binding, which results in translation being inhibited at the level of cap recognition. As a result of bypassing the need to disrupt the very stable eIF4E:eIF4G interaction, d4EHP should interdict translation more efficiently than 4E-BPs or other eIF4E binding proteins. 4EHP-mediated translational regulation may have a particularly important role in germline development, based on these results and on a recent report that a mutant allele of C. elegans 4EHP (ife-4) shows a severe egg-laying defect (Cho, 2005).

The delineation of a d4EHP-recognition sequence in Bcd (YxxxxxxL; x denotes any amino acid) that interacts with d4EHP via its Trp85 residue highlights the similarities between the d4EHP:Bcd interaction and that of eIF4G with eIF4E (YxxxxLphi in eIF4G; Trp73 in eIF4E; phi denotes any hydrophobic amino acid). Despite these parallels, the inability of Bcd to bind to eIF4E must be explained by structural differences. The presence of two proline residues at position +3 and +6 of the Bcd d4EHP binding motif is predicted to significantly alter the α-helical structure assumed by the YxxxxLphi peptide upon binding to eIF4E and thus prevent Bcd association with deIF4E. Furthermore, the eIF4E interaction surface of eIF4G is not limited to the YxxxxLphi motif but extends over a larger interface; the N-terminal domain of eIF4E is also required for folding and tight binding to eIF4G. Indeed, the ability of d4EHP to bind specifically to Bcd, and not to deIF4G and d4E-BP, can be explained by the importance of the N-terminal KHPL sequence of eIF4E in the interaction with eIF4G and 4E-BP, since this sequence is not conserved in d4EHP (Cho, 2005).

The demonstration that cad translation is repressed through a d4EHP- and Bcd-dependent tethering mechanism adds to the diversity of translational control mechanisms operating in the early Drosophila embryo. Why are so many translational repression pathways necessary? If an individual mechanism alone can reduce translation of a specific mRNA, but not completely abrogate it, a combination of inhibitory interactions may be needed in order to accomplish strict translational control. This can be advantageous if the diversity of factors (like Bcd, which can confer mRNA specificity for a given mechanism) is relatively limited. Multiple mRNAs also have to be translationally repressed in overlapping spatial and temporal domains. Controlling these mRNAs through mechanisms that target different components of the general translational machinery, rather than through a common mechanism, might allow more precise regulation of their individual expression patterns (Cho, 2005).

It is noteworthy that although 4EHP is conserved through evolution, Bcd exists only in higher dipterans. Thus, in other organisms, 4EHP must function during development through proteins that are analogous to Bcd. In summary, this study describes a novel mode of translational control in Drosophila development. Because cap-dependent translation regulation plays such an important role in gene expression, and since 4EHP is also expressed in somatic cells, it is predicted that examples of d4EHP-mediated translational repression other than cad are most likely to exist (Cho, 2005).

eIF4A inactivates TORC1 in response to amino acid starvation

Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. This study identified the the eIF4A-containing eIF4F translation initiation complex (composed of three subunits: eIF4E, eIF4A and eIF4G) as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal (Tsokanos, 2016).

To maintain homeostasis, biological systems frequently use a combination of two distinct mechanisms that converge and counteract each other. For instance, the level of phosphorylation of a target protein depends not only on the rate of phosphorylation by the upstream kinase, but also on the rate of dephosphorylation by the phosphatase. Both the activating kinase and the inactivating phosphatase can be regulated separately. Likewise, the activity of TORC1 in response to amino acid levels appears to reflect a balance between activating and inactivating mechanisms that converge on Rheb. When amino acids are re-added to cells, TORC1 is activated via Rag or Arf1 GTPase-dependent recruitment to the lysosome where TORC1 binds Rheb. In contrast, when amino acids are removed from cells, TORC1 activity drops in part by blocking this activation mechanism and in part via a distinct inactivation mechanism whereby TSC2 is recruited to the vicinity of TORC1 to act on Rheb (Demetriades, 2014). The existence of this distinct and counteracting mechanism is highlighted by the fact that in the absence of TSC2, both Drosophila and mammalian cells do not appropriately inactivate TORC1 in response to amino acid removal. The upstream mechanisms regulating TSC2 in response to amino acid withdrawal, however, are not known. This study has identified the translational machinery, and in particular components of the eIF4F complex, as one upstream regulatory mechanism working via TSC2 to inactivate TORC1 upon amino acid withdrawal (Tsokanos, 2016).

The subcellular localization of TORC1 plays an important role in its regulation. A significant body of evidence shows that TORC1 needs to translocate to the lysosome or Golgi to become reactivated following amino acid starvation and re-addition. Whether active TORC1 then remains on the lysosome, or whether it can move elsewhere in the cell to phosphorylate target proteins, is less clear. Several findings in the literature, as well as the data presented in this study, indicate that active TORC1 can leave the lysosome, yet remain active: (1) Upon amino acid re-addition in starved cells, the Rag GTPases are necessary for mTORC1 lysosomal localization and reactivation. In contrast, Rag depletion in cells growing under basal conditions, replete of serum and amino acids, does not cause a strong drop in mTORC1 activity, although it causes a similar delocalization of mTORC1 away from lysosomes. Hence, under these conditions, mTORC1 is non-lysosomal, but still active to a large extent. (2) Similarly, particular stresses such as arsenite treatment can cause TORC1 to localize away from the lysosome, yet remain active. (3) The Rag GTPases tether TORC1 to the LAMTOR complex present on the lysosome. Amino acid restimulation, which activates TORC1, actually decreases binding between Rag GTPases and LAMTOR, suggesting that active Rag-bound TORC1 complexes can leave the lysosome and reside elsewhere in the cell. Additional mechanisms also contribute to the delocalization of the Rag GTPases away from lysosomes (4) Active TORC1 phosphorylates target proteins such as 4E-BP and S6K, which are physically associated with translation preinitiation complexes. Indeed, this study reports physical interactions between the TORC1 complex and translation preinitiation complexes, in agreement with what has also been observed by others. Therefore, either translation preinitiation complexes need to translocate to lysosomes to meet TORC1, or TORC1 needs to come off the lysosome to meet translation preinitiation complexes in the cytoplasm. (5) Using proximity ligation assay, an interaction was observed between Raptor and eIF4A, which does not colocalize with either lysosomes or endoplasmic reticulum, suggesting that it takes place in the cytoplasm. (6) In agreement with these PLA data, antibody staining of cells in the presence of amino acids with anti-TOR antibody reveals an accumulation of TOR on lysosomes, as well as a more diffuse, non-lysosomal TORC1 localization throughout the cytoplasm. (7) A recent report employing a FRET-based probe detects mTORC1 activity at lysosomes as well as in the cytoplasm and nucleus. Taken together, these data suggest that although TORC1 is activated on the lysosome, it then in part translocates to other sites in the cell including the cytoplasm to phosphorylate target proteins (Tsokanos, 2016).

Upon amino acid withdrawal, both cytoplasmic and lysosomal fractions of active TORC1 need to be inactivated. The data presented in this study suggest that upon amino acid removal, inactivation of TORC1 happens in part via an eIF4A-dependent mechanism acting on TSC2 to inactivate Rheb in the cytosol. In agreement with this, TORC1 inactivation upon amino acid removal can be rescued by supplying cells with dominantly active, but not wild-type Rheb. It has been previously reported that a pool of TSC2 is also recruited to lysosomes upon amino acid removal. This study shows in Drosophila cells, upon amino acid removal, some TSC2 accumulates in lysosomes, whereas some remains in the cytosol. Therefore, TSC2 is likely recruited to all subcellular sites where active TORC1 is located to inactivate it. Indeed, Rheb and TSC2 have been observed at several subcellular compartments. Since Rheb localizes to many endomembranes in the cell, Rheb that is not bound to TORC1 could potentially remain active, to provide a pool for subsequent TORC1 reactivation (Tsokanos, 2016).

Upon inactivation, the data indicate that TORC1 remains bound to preinitiation complexes, in agreement with previous reports. This finding is reminiscent of the fact that Raptor is also recruited to stress granules, which are essentially stalled preinitiation complexes, in response to another stress-oxidative stress. Whether the Rag GTPases also remain bound to preinitiation complexes upon amino acid removal is unclear because some experiments showed a decrease in binding between Rag GTPases and initiation factors, and some did not (Tsokanos, 2016).

How could eIF4A affect TORC1 activity? The data indicate that the effects of eIF4A knockdown cannot be explained as a consequence of generally impaired translation, since other means of blocking translation do not have the same effects on TORC1 activity upon amino acid starvation. Instead, knockdown of any of the three members of the eIF4F complex gives this elevated TORC1 phenotype, indicating that it is specific for the eIF4F complex. The data are consistent with two interpretations: One option is that the eIF4F complex is specifically required to translate a protein that promotes TSC2 function. An alternate option is that the eIF4F complex acts directly on TSC2, regulating its activity. The latter is supported by the fact that eIF4A and TSC2 proteins are seen interacting with each other. Interestingly, eIF4A has been reported to have additional functions that are not translation-related (Tsokanos, 2016).

Some differences were noted between Drosophila cells and mammalian cells. The first is that overexpression of wild-type Rheb is sufficient to activate TORC1 upon amino acid removal in mammalian cells, whereas this is not the case in Drosophila cells. This could be due to a difference in the biology of the two cell types, or simply to a technical difference having to do with levels of Rheb overexpression. A second difference is that cycloheximide treatment is sufficient to maintain elevated TORC1 levels in HeLa or HEK293 cells upon amino acid removal, whereas this is not the case in Drosophila cells. This could be due to differences in rates of amino acid efflux and levels of autophagy in mammalian compared to S2 and Kc167 cells, causing intracellular amino acid levels to remain elevated in mammalian cells when both amino acid import from the medium and amino acid expenditure via translation are simultaneously blocked (Tsokanos, 2016).

A number of studies have looked at the involvement of Rheb in the cellular response to amino acids, with some disagreement on whether amino acids affect Rheb GTP-loading or Rheb-mTOR binding. The current data fit with previous reports that Rheb GTP-loading is affected by amino acids and with the conclusion that amino acids affect TORC1 activity via both a Rheb-dependent and a Rheb-independent mechanism (Tsokanos, 2016).

The data indicate a close physical relationship between TORC1 and the translational machinery. This is in part mediated by a direct interaction between the major scaffolding subunit of the initiation complex, eIF4G, and RagC and in part likely mediated by additional interactions between TORC1 and preinitiation supercomplexes as previously reported. Interestingly, TORC2 is also physically associated with the ribosome and requires ribosomes, but not translation, for its activation. Hence, both TORC1 and TORC2 have close physical connections to the translational machinery (Tsokanos, 2016).

Some side observations in this study are interesting and could constitute a starting point for further studies. For instance, eIF4A-knockdown cells inactivate TORC1 more robustly than control cells upon serum removal. Also, eIF2b knockdown causes S6K phosphorylation to decrease significantly in S2 cells. It is not known why this occurs. The latter might suggest that there are additional points of cross-talk between TORC1 and the translation machinery (Tsokanos, 2016).

How cells sense the presence or the absence of amino acids has been an open question in the field. The data presented in this study indicate that the translational machinery itself might sense the absence of amino acids. Indeed, the relevant parameter for a cell is likely not the absolute levels of intracellular amino acids, but rather whether the available amino acid levels are sufficient to support the amount of translation that a cell requires. Hence, the translation machinery itself might be best poised to make this assessment. Binding is observed between eIF4A and NAT1 that is strong in the presence of amino acids, and is reduced upon amino acid withdrawal, independently of TORC1 signaling. These epistasis experiments are consistent with NAT1 acting as the upstream mediator of the amino acid signal, binding and inhibiting eIF4A in the presence of amino acids, but not in the absence of amino acids. Hence, NAT1 might play a role in this sensing process (Tsokanos, 2016).

In sum, these data identify the eIF4F complex as an important upstream regulator of TORC1, which acts via TSC2 to inactivate TORC1 upon withdrawal of amino acids (Tsokanos, 2016).

Search PubMed for articles about Drosophila 4EHP

Chagnovich, D. and Lehmann, R. (2001). Poly(A)-independent regulation of maternal hunchback translation in the Drosophila embryo. Proc. Natl. Acad. Sci. 98: 11359-64. PubMed ID: 11562474

Chekulaeva, M., Hentze, M. W. and Ephrussi, A. (2006). Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles. Cell 124: 521-33. PubMed ID: 16469699

Cho, P. F., et al. (2005). A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP. Cell 121(3): 411-23. PubMed ID; Online text

Cho, P. F., et al. (2006). Cap-dependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos. Curr. Biol. 16(20): 2035-41. PubMed ID: 17055983

Edwards, T. A., et al. (2003). Model of the Brain tumor-Pumilio translation repressor complex. Genes Dev. 17: 2508-2513. PubMed ID: 14561773

Niessing, D., Blanke, S. and Jackle, H. (2002). Bicoid associates with the 5'-cap-bound complex of caudal mRNA and represses translation. Genes Dev. 16(19): 2576-82. PubMed ID: 12368268

Sonoda, J. and Wharton, R. P. (2001). Drosophila Brain Tumor is a translational repressor. Genes Dev. 15: 762-73. PubMed ID: 11274060

Tsokanos, F. F., Albert, M. A., Demetriades, C., Spirohn, K., Boutros, M. and Teleman, A. A. (2016). eIF4A inactivates TORC1 in response to amino acid starvation. EMBO J 35(10):1058-76. PubMed ID: 26988032

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date revised: 15 December 2019

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