Martin D. Jennings

eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation

In protein synthesis initiation, the eukaryotic translation initiation factor (eIF) 2 (a G protein) functions in its GTP-bound state to deliver initiator methionyl-tRNA (tRNAiMet) to the small ribosomal subunit and is necessary for protein synthesis in all cells. Phosphorylation of eIF2 [eIF2(αP)] is critical for translational control in diverse settings including nutrient deprivation, viral infection and memory formation. eIF5 functions in start site selection as a GTPase accelerating protein (GAP) for the eIF2·GTP·tRNAiMet ternary complex within the ribosome-bound pre-initiation complex. Here we define new regulatory functions of eIF5 in the recycling of eIF2 from its inactive eIF2·GDP state between successive rounds of translation initiation. First we show that eIF5 stabilizes the binding of GDP to eIF2 and is therefore a bi-functional protein that acts as a GDP dissociation inhibitor (GDI). We find that this activity is independent of the GAP function and identify conserved residues within eIF5 that are necessary for this role. Second we show that eIF5 is a critical component of the eIF2(αP) regulatory complex that inhibits the activity of the guanine-nucleotide exchange factor (GEF) eIF2B. Together our studies define a new step in the translation initiation pathway, one that is critical for normal translational controls.

eIF2B promotes eIF5 dissociation from eIF2•GDP to facilitate guanine nucleotide exchange for translation initiation

Protein synthesis factor eIF2 delivers initiator tRNA to the ribosome. Two proteins regulate its G-protein cycle: eIF5 has both GTPase-accelerating protein (GAP) and GDP dissociation inhibitor (GDI) functions, and eIF2B is the guanine nucleotide exchange factor (GEF). In this study, we used protein-protein interaction and nucleotide exchange assays to monitor the kinetics of eIF2 release from the eIF2•GDP/eIF5 GDI complex and determine the effect of eIF2B on this release. We demonstrate that eIF2B has a second activity as a GDI displacement factor (GDF) that can recruit eIF2 from the eIF2•GDP/eIF5 GDI complex prior to GEF action. We found that GDF function is dependent on the eIF2Bε and eIF2Bγ subunits and identified a novel eIF2-eIF2Bγ interaction. Furthermore, GDF and GEF activities are shown to be independent. First, eIF2B GDF is insensitive to eIF2α phosphorylation, unlike GEF. Second, we found that eIF2Bγ mutations known to disrupt GCN4 translational control significantly impair GDF activity but not GEF function. Our data therefore define an additional step in the protein synthesis initiation pathway that is important for its proper control. We propose a new model to place eIF2B GDF function in the context of efficient eIF2 recycling and its regulation by eIF2 phosphorylation.

eIF2B is a decameric guanine nucleotide exchange factor with a γ2ε2 tetrameric core

eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (α–ε), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic γ- and ε-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2Bγ, prompting us to propose a multi-step mechanism for nucleotide exchange.

Specificity and autoregulation of notch binding by tandem WW domains in suppressor of deltex.

WW domains target proline-tyrosine (PY) motifs and frequently function as tandem pairs. When studied in isolation, single WW domains are notably promiscuous and regulatory mechanisms are undoubtedly required to ensure selective interactions. Here, we show that the fourth WW domain (WW4) of Suppressor of Deltex, a modular Nedd4-like protein that down-regulates the Notch receptor, is the primary mediator of a direct interaction with a Notch-PY motif. A natural Trp to Phe substitution in WW4 reduces its affinity for general PY sequences and enhances selective interaction with the Notch-PY motif via compensatory specificity-determining interactions with PY-flanking residues. When WW4 is paired with WW3, domain-domain association, impeding proper folding, competes with Notch-PY binding to WW4. This novel mode of autoinhibition is relieved by binding of another ligand to WW3. Such cooperativity may facilitate the transient regulatory interactions observed in vivo between Su(dx) and Notch in the endocytic pathway. The highly conserved tandem arrangement of WW domains in Nedd4 proteins, and similar arrangements in more diverse proteins, suggests domain-domain communication may be integral to regulation of their associated cellular activities.

Su(dx) E3 ubiquitin ligase–dependent and –independent functions of Polychaetoid, the Drosophila ZO-1 homologue

Polychaetoid coordinates receptor trafficking and signaling with adherens junction organization.

eIF5: A dual function GAP and GDI for eukaryotic translational control

We recently showed in a publication in Nature that the eukaryotic translation initiation factor eIF5 has a second regulatory function and is a GDI (GDP dissociation inhibitor) in addition to its previously characterized role as a GAP (GTPase accelerating protein). These findings provide new insight into the mechanism of translation initiation in eukaryotic cells. Additional findings show that the GDI function is critical for the normal regulation of protein synthesis by phosphorylation of eIF2α at ser51. Because eIF2 phosphorylation is a ubiquitous mode of translational control these results are of broad interest. Here we review these and related studies and suggest they offer further evidence of parallels between the functions of regulators of the translation factor eIF2 and both heterotrimeric and small GTPases.

Clues to the mechanism of action of eIF2B, the guanine-nucleotide-exchange factor for translation initiation

A variety of cellular processes rely on G-proteins, which cycle through active GTP-bound and inactive GDP-bound forms. The switch between these states is commonly regulated by GEFs (guanine-nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Although G-proteins have structural similarity, GEFs are very diverse proteins. A complex example of this system is seen in eukaryotic translation initiation between eIF (eukaryotic initiation factor) 2, a G-protein, its five-subunit GEF, eIF2B, and its GAP, eIF5. eIF2 delivers Met-tRNA(i) (initiator methionyl-tRNA) to the 40S ribosomal subunit before mRNA binding. Upon AUG recognition, eIF2 hydrolyses GTP, aided by eIF5. eIF2B then re-activates eIF2 by removing GDP, thereby promoting association of GTP. In the present article, we review data from studies of representative G-protein-GEF pairs and compare these with observations from our research on eIF2 and eIF2B to propose a model for how interactions between eIF2B and eIF2 promote guanine nucleotide exchange.

Mechanisms of translational regulation by a human eIF5-mimic protein

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNAiMet ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

A new function and complexity for protein translation initiation factor eIF2B

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.

Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes

Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B. DOI: http://dx.doi.org/10.7554/eLife.24542.001