Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome

eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner (K_i^{60S-eIF5A-Hyp} = 16 nM, K_i^{60S-eIF5A-Lys} = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon charged-tRNA_i^Met binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5A^Q22H/L93F and eIF5A^K56A, display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5A^K56A, but not eIF5A^Q22H/L93F, is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5A^Q22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects.     

Bestrophin interacts physically and functionally with protein phosphatase 2A

Bestrophin is a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial cells (RPE). It is encoded by the VMD2 gene, which is mutated in Best macular dystrophy, a disease characterized by a depressed light peak in the electrooculogram. Recently it was proposed that bestrophin is a chloride channel responsible for generating the light peak. To investigate its function further, we immunoaffinity purified a bestrophin complex from RPE lysates and identified bestrophin and the beta-catalytic subunit of protein phosphatase 2A (PP2A) as members of the complex by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Protein-protein interaction between bestrophin and PP2Ac and the structural subunit of PP2A, PR65, was confirmed by reciprocal immunoprecipitation. The C-terminal cytoplasmic domain of bestrophin was sufficient for the interaction with PP2A as demonstrated by a pulldown assay using a fusion of this domain with glutathione S-transferase. Bestrophin was phosphorylated when expressed in RPE-J cells and this phosphorylation was sensitive to okadaic acid. Purified PP2A effectively dephosphorylated bestrophin in vitro. These data suggest that bestrophin is in the signal transduction pathway that modulates the light peak of the electrooculogram, that it is regulated by phosphorylation, and that phosphorylation of bestrophin is in turn regulated by PP2A.     

CHIP/Stub1 interacts with eIF5A and mediates its degradation

eIF5A, containing the unusual amino acid hypusine, is a highly conserved protein essential for the proliferation of eukaryotic cells. Previous studies have demonstrated that the activity of eIF5A was regulated through modification of hypusine, phosphorylation and acetylation. However, no study was documented for regulation of the protein stability. Here, we report that eIF5A is a target of CHIP (the carboxyl terminus of Hsc70-interacting protein, also named Stub1), an E3 ligase with a U-box domain, through a proteomics analysis. CHIP directly interacted with eIF5A, preferably through the U-box domain, to mediate eIF5A ubiquitination and degradation. Simultaneously, we investigated that CHIP expression inversely correlated with eIF5A levels in colorectal cancers, consistent with the fact that the protein level of eIF5A was increased in the CHIP knock-out MEF cells. Taken together, we propose that CHIP regulates the eIF5A protein stability via a protein degradation mechanism. This study provides a new insight into understanding the regulation of the eIF5A stability.     

The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex

Dcp1 plays a key role in the mRNA decay process in Saccharomyces cerevisiae, cleaving off the 5' cap to leave an end susceptible to exonucleolytic degradation. The eukaryotic initiation factor complex eIF4F, which in yeast contains the core components eIF4E and eIF4G, uses the cap as a binding site, serving as an initial point of assembly for the translation apparatus, and also binds the poly(A) binding protein Pab1. We show that Dcp1 binds to eIF4G and Pab1 as free proteins, as well as to the complex eIF4E-eIF4G-Pab1. Dcp1 interacts with the N-terminal region of eIF4G but does not compete significantly with eIF4E or Pab1 for binding to eIF4G. Most importantly, eIF4G acts as a function-enhancing recruitment factor for Dcp1. However, eIF4E blocks this effect as a component of the high affinity cap-binding complex eIF4E-eIF4G. Indeed, cooperative enhancement of the eIF4E-cap interaction stabilizes yeast mRNAs in vivo. These data on interactions at the interface between translation and mRNA decay suggest how events at the 5' cap and 3' poly(A) tail might be coupled.     

Further evidence that ribavirin interacts with eIF4E

This commentary discusses the recent reports in RNA by Yan and colleagues and Westman and colleagues of the apparent failure of ribavirin to bind to recombinant eIF4E and inhibit 7-methyl guanosine cap-dependent exogenous mRNA translation of cell extracts in vitro. Measuring binding by using affinity chromatography of matrix-immobilized proteins and by using protein emission fluorescence spectroscopy in the presence of nucleotide ligands, as well as limitations of using cell extracts for the assessment of mechanisms of mRNA translation are discussed. Possible reasons for the discordant findings of Yan and colleagues and Westman and colleagues are suggested, and direct observation of the specific binding of ribavirin to eIF4E by using mass spectrometry is presented.     

4E-BP3 regulates eIF4E-mediated nuclear mRNA export and interacts with replication protein A2

In nucleus, eIF4E regulates the nucleus export of specific mRNA. In this study, altered 4E-BP3 (eIF4E-binding protein 3) expression resulted in profoundly affected cyclin D1 protein levels, partially due to changes in the cytoplasmic cyclin D1 mRNA levels in both U2OS and MCF7 cells, whereas altered 4E-BP1 expression did not affect eIF4E-mediated cyclin D1 mRNA export. 4E-BP3 also affected a subset of growth promoting mRNAs exported in an eIF4-dependent manner. Furthermore, 4E-BP3 interacted with dephosphorylated RPA2 (replication protein A2). The results indicated 4E-BP3 acts as an inhibitor of eIF4E-mediated mRNA export in the examined cells, and 4E-BP3 inhibition of eIF4E-mediated mRNA export is regulated by the phosphorylation state of RPA2.     

Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.     

The yeast eukaryotic initiation factor 4G (eIF4G) HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection

Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel alpha-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, co-overexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts(-) mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by co-overexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.     

Molecular docking uncovers TSPY binds more efficiently with eEF1A2 compared to eEF1A1

Testis-specific protein, Y-encoded (TSPY) binds to eukaryotic translation elongation factor 1 alpha (eEF1A) at its SET/NAP domain that is essential for the elongation during protein synthesis implicated with normal spermatogenesis. The eEF1A exists in two forms, eEF1A1 (alpha 1) and eEF1A2 (alpha 2), encoded by separate loci. Despite critical interplay of the TSPY and eEF1A proteins, literature remained silent on the residues playing significant roles during such interactions. We deduced 3D structures of TSPY and eEF1A variants by comparative modeling (Modeller 9.13) and assessed protein–protein interactions employing HADDOCK docking. Pairwise alignment using EMBOSS Needle for eEF1A1 and eEF1A2 proteins revealed high degree (~92%) of homology. Efficient binding of TSPY with eEF1A2 as compared to eEF1A1 was observed, in spite of the occurrence of significant structural similarities between the two variants. We also detected strong interactions of domain III followed by domains II and I of both eEF1A variants with TSPY. In the process, seven interacting residues of TSPY’s NAP domain namely, Asp 175, Glu 176, Asp 179, Tyr 183, Asp 240, Glu 244, and Tyr 246 common to both eEF1A variants were detected. Additionally, six lysine residues observed in eEF1A2 suggest their possible role in TSPY–eEF1A2 complex formation essential for germ cell development and spermatogenesis. Thus, more efficient binding of TSPY with eEF1A2 as compared to that of eEF1A1 established autonomous functioning of these two variants. Studies on mutated protein following similar approach would uncover the causative obstruction, between the interacting partners leading to deeper understanding on the structure–function relationship.     

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

In eukaryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2.GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bvarepsilon catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bvarepsilon mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bvarepsilon with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bvarepsilon segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bvarepsilon to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNA(i)(Met), suggesting that tRNA(i)(Met) is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation.     

Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo

Translation initiation factor 1A (eIF1A) is predicted to bind in the decoding site of the 40S ribosome and has been implicated in recruitment of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and ribosomal scanning. We show that the unstructured C-terminus of eIF1A interacts with the C-terminus of eIF5B, a factor that stimulates 40S-60S subunit joining, and removal of this domain of eIF1A diminishes translation initiation in vivo. These findings support the idea that eIF1A-eIF5B association is instrumental in releasing eIF1A from the ribosome after subunit joining. A larger C-terminal truncation that removes a 3(10) helix in eIF1A deregulates GCN4 translation in a manner suppressed by overexpressing TC, implicating eIF1A in TC binding to 40S ribosomes in vivo. The unstructured N-terminus of eIF1A interacts with eIF2 and eIF3 and is required at low temperatures for a step following TC recruitment. We propose a modular organization for eIF1A wherein a core ribosome-binding domain is flanked by flexible segments that mediate interactions with other factors involved in recruitment of TC and release of eIF1A at subunit joining.     

Interaction of recombinant human eIF2 subunits with eIF2B and eIF2alpha kinases

The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2alpha kinase, is found to interact with wt and mutants of eIF2alpha in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2alpha. The dissociation constant of alpha-subunit-PERK complex (Kd alpha-subunit) is 0.74 microM and the interaction is stoichiometric.     

Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. eIF5A-1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A-2 mRNA (0.7-5.6 kb) and eIF5A-2 protein (20 kDa) varies widely. Whereas eIF5A-2 mRNA was demonstrable in most cells, eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high overexpression of eIF5A-2 mRNA. Multiple forms of eIF5A-2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3'-UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A-1 and eIF5A-2 precursor proteins were modified comparably in UACC-1598 cells and both were similarly stable. When eIF5A-1 and eIF5A-2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A-2 precursor was synthesized at a level comparable to that of eIF5A-1 precursor, indicating that the elements causing inefficient translation of eIF5A-2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A-2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A-1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A-2 protein even in eIF5A-2 mRNA positive cells is, at least in part, due to inefficient translation.     

A novel protein interacts with the Werner's syndrome gene product physically and functionally

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.     

Cloning and characterization of the p42 subunit of mammalian translation initiation factor 3 (eIF3): demonstration that eIF3 interacts with eIF5 in mammalian cells.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit protein complex that plays an essential role in the binding of the initiator methionyl-tRNA and mRNA to the 40S ribosomal subunit to form the 40S initiation complex. cDNAs encoding all the subunits of mammalian eIF3 except the p42 subunit have been cloned in several laboratories. Here we report the cloning and characterization of a human cDNA encoding the p42 subunit of mammalian eIF3. The open reading frame of the cDNA, which encodes a protein of 320 amino acids (calculated Mr35 614) has been expressed in Escherichia coli and the recombinant protein has been purified to homogeneity. The purified protein binds RNA in agreement with the presence of a putative RNA binding motif in the deduced amino acid sequence. The protein shows 33% identity and 53% similarity with the Tif35p subunit (YDR 429C) of yeast eIF3. Transfection experiments demonstrated that polyhistidine-tagged p42 protein, transiently expressed in human U20S cells, was incorporated into endogenous eIF3. Furthermore, eIF3 isolated from transfected cell lysates contains bound eIF5 indicating that a specific physical interaction between eIF5 and eIF3 may play an important role in the function of eIF5 during translation initiation in eukaryotic cells.     

Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-beta-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.