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Umadas Maitra - One of the best experts on this subject based on the ideXlab platform.

  • casein kinase ii phosphorylates translation Initiation Factor 5 eif5 in saccharomyces cerevisiae
    Yeast, 2003
    Co-Authors: Tapan Maiti, Amitabha Bandyopadhyay, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts with the 40S Initiation complex (40S-eIF3-mRNA-Met-tRNA(f)-eIF2-GTP) to promote the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single-copy essential gene, TIF5, that is required for cell growth and viability. In this work, we show that eIF5 immunoprecipitated from cell-free extracts of (32)P-labelled yeast cells is phosphorylated on multiple serine residues. Phosphopeptide mapping reveals four major sites of phosphorylation that appear to be identical to recombinant yeast eIF5 sites phosphorylated in vitro by casein kinase II. Furthermore, analysis of eIF5 isolated from a yeast strain having a conditional mutant of casein kinase II indicates that phosphorylation of eIF5 is completely abolished at the non-permissive temperature. Additionally, haploid yeast strains were constructed to contain Ser-to-Ala mutations at the five casein kinase II consensus sequences in eIF5; in these cells, eIF5 phosphorylation was absent. Surprisingly, substitution of the TIF5 gene mutated at these sites for the wild-type gene had no obvious effect on cell growth under normal growth conditions. The implications of these results in eIF5 function are discussed.

  • phosphorylation of mammalian translation Initiation Factor 5 eif5 in vitro and in vivo
    Nucleic Acids Research, 2002
    Co-Authors: Romit Majumdar, Amitabha Bandyopadhyay, Haiteng Deng, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts with the 40S Initiation complex (40S•eIF3•AUG•Met-tRNAf•eIF2•GTP) and, acting as a GTPase activating protein, promotes the hydrolysis of bound GTP. We isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate purified bacterially expressed recombinant rat eIF5. Physical, biochemical and antigenic properties of this kinase identify it as casein kinase II (CK II). Mass spectrometric analysis of maximally in vitro phosphorylated eIF5 localized the major phosphorylation sites at Ser-387 and Ser-388 near the C-terminus of eIF5. These serine residues are embedded within a cluster of acidic amino acid residues and account for nearly 90% of the total in vitro eIF5 phosphorylation. A minor phosphorylation site at Ser-174 was also observed. Alanine substitution mutagenesis at Ser-387 and Ser-388 of eIF5 abolishes phosphorylation by the purified kinase as well as by crude reticulocyte lysates. The same mutations also abolish phosphorylation of eIF5 when transfected into mammalian cells suggesting that CK II phosphorylates eIF5 at these two serine residues in vivo as well.

  • Eukaryotic Translation Initiation Factor 5 Functions as a GTPase-activating Protein
    The Journal of biological chemistry, 2000
    Co-Authors: Supratik Das, Rajarshi Ghosh, Umadas Maitra
    Abstract:

    Abstract Eukaryotic translation Initiation Factor 5 (eIF5) forms a complex with eIF2 by interacting with the β subunit of eIF2. This interaction is essential for eIF5-promoted hydrolysis of GTP bound to the 40 S Initiation complex. In this work, we show that, in addition to the eIF2β-binding region at the C terminus of eIF5, the N-terminal region of eIF5 is also required for eIF5-dependent GTP hydrolysis. Like other GTPase-activating proteins, eIF5 contains an invariant arginine residue (Arg-15) at its N terminus that is essential for its function. Mutation of this arginine residue to alanine or even to conservative lysine caused a severe defect in the ability of eIF5 to promote GTP hydrolysis from the 40 S Initiation complex, although the ability of these mutant proteins to bind to eIF2β remained unchanged. These mutants were also defective in overall protein synthesis as well as in their ability to support cell growth of a ΔTIF5yeast strain. Additionally, alanine substitution mutagenesis of eIF5 defined Lys-33 and Lys-55 as also critical for eIF5 function in vitro and in vivo. The implications of these results in relation to other well characterized GAPs are discussed and provide additional evidence that eIF5 functions as a GTPase-activating protein.

  • mutational analysis of mammalian translation Initiation Factor 5 eif5 role of interaction between the β subunit of eif2 and eif5 in eif5 function in vitro and in vivo
    Molecular and Cellular Biology, 2000
    Co-Authors: Supratik Das, Umadas Maitra
    Abstract:

    Eukaryotic Initiation Factor 5 (eIF5), a monomeric protein of 49 kDa in mammals (9, 10, 21) and 46 kDa in the yeast Saccharomyces cerevisiae (5, 6), plays an essential role in the Initiation of protein synthesis. Following scanning of mRNA by the 40S preInitiation complex (40S–eIF3–Met-tRNAf–eIF2–GTP) and positioning of the initiator Met-tRNAf at the AUG codon of the mRNA to form the 40S Initiation complex (eIF3–40S–AUG–Met-tRNAf–eIF2–GTP), the Initiation Factor eIF5 interacts with the 40S Initiation complex to effect the hydrolysis of ribosome-bound GTP. Hydrolysis of GTP causes the release of eIF2-GDP, Pi, and eIF3 from the 40S Initiation complex, which is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form a functional 80S Initiation complex (80S–mRNA–Met-tRNAf) that is active in peptidyl transfer (for reviews, see references 16, 18, and 19). eIF5-dependent GTP hydrolysis has also been shown to play an important role in the selection of the AUG start codon (15). An interesting feature of the derived amino acid sequence of mammalian (rat and human) and yeast eIF5 proteins (26) is the presence of sequence motifs at the N-terminal region of eIF5 that have weak homology to characteristic domains present in proteins belonging to the GTPase superfamily (3). However, unlike these proteins, eIF5 neither binds nor hydrolyzes free GTP or GTP bound to the (Met-tRNAf–eIF2–GTP) ternary complex in the absence of 40S ribosomal subunits (4, 7). eIF5 promotes GTP hydrolysis only when the nucleotide is bound to eIF2 in the 40S Initiation complex (Met-tRNAf–eIF2–GTP–eIF3–40S–AUG) (4, 7). These results suggest that eIF5 must interact with one or more components of the 40S Initiation complex to cause hydrolysis of GTP. In agreement with this hypothesis, we observed that mammalian eIF5 forms a complex with mammalian eIF2 (7), a component of the 40S Initiation complex, and that eIF5-eIF2 complex formation occurs through the β subunit of eIF2 (11). Complex formation between eIF5 and Nip1p subunit of eIF3 has also been reported (1, 2). In the case of eIF5 and eIF2β interaction, deletion studies have shown that the N-terminal region of eIF2β binds eIF5 and that the conserved stretch of lysine residues in this region plays an important role in this interaction (11). Similar interaction between yeast eIF5 and yeast eIF2β was also reported from this laboratory (11) and later by others (1), indicating that the interaction domains of eIF5 and the β subunit of eIF2 are conserved through evolution. In later studies, Asano et al. (1) observed that a bipartite motif at the C-terminal region of yeast eIF5 containing conserved aromatic and acidic residues is required for binding to both eIF2β and the Nip1p subunit of eIF3. However, the important question remained as to whether the interaction of eIF5 with eIF2β is required for eIF5-dependent GTP hydrolysis and is thus a key molecular interaction in the translation Initiation pathway. In the work presented here, we demonstrate that mammalian eIF5 interacts with mammalian eIF2β through a conserved C-terminal region. We have carried out a systematic mutational analysis of conserved residues in the C-terminal eIF2β-binding region of rat eIF5 to generate mutants which are defective in binding to eIF2β but are active in binding to Nip1p. We show that these mutants are also defective in eIF5-dependent GTP hydrolysis and consequently in 80S Initiation complex formation as well as in in vitro protein synthesis. Furthermore, whereas mammalian eIF5 can functionally substitute for the homologous yeast protein in vivo in yeast cells (17), the mutant eIF5 proteins that are defective in binding to eIF2β are unable to complement a genetic disruption in the chromosomal copy of the TIF5 gene in vivo. Taken together, our results suggest that the interaction between eIF5 and the β subunit of eIF2 is required for eIF5-dependent hydrolysis of GTP during translation Initiation and consequently is essential for overall protein synthesis.

  • specific interaction of eukaryotic translation Initiation Factor 5 eif5 with the β subunit of eif2
    Journal of Biological Chemistry, 1997
    Co-Authors: Supratik Das, Kallol Das, Tapan Maiti, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts with the 40 S Initiation complex (40 S·mRNA· eIF3·Met-tRNAf·eIF2·GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude Initiation Factor preparations. We observed that eIF5 specifically interacted with the β subunit of Initiation Factor eIF2. No other Initiation Factors including the γ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the β subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2β deletion mutants expressed inEscherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2β. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf·eIF2·GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the β subunit of eIF2 may be critical for the hydrolysis of GTP during translation Initiation.

Lunet E Luna - One of the best experts on this subject based on the ideXlab platform.

Tapan Maiti - One of the best experts on this subject based on the ideXlab platform.

  • casein kinase ii phosphorylates translation Initiation Factor 5 eif5 in saccharomyces cerevisiae
    Yeast, 2003
    Co-Authors: Tapan Maiti, Amitabha Bandyopadhyay, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts with the 40S Initiation complex (40S-eIF3-mRNA-Met-tRNA(f)-eIF2-GTP) to promote the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single-copy essential gene, TIF5, that is required for cell growth and viability. In this work, we show that eIF5 immunoprecipitated from cell-free extracts of (32)P-labelled yeast cells is phosphorylated on multiple serine residues. Phosphopeptide mapping reveals four major sites of phosphorylation that appear to be identical to recombinant yeast eIF5 sites phosphorylated in vitro by casein kinase II. Furthermore, analysis of eIF5 isolated from a yeast strain having a conditional mutant of casein kinase II indicates that phosphorylation of eIF5 is completely abolished at the non-permissive temperature. Additionally, haploid yeast strains were constructed to contain Ser-to-Ala mutations at the five casein kinase II consensus sequences in eIF5; in these cells, eIF5 phosphorylation was absent. Surprisingly, substitution of the TIF5 gene mutated at these sites for the wild-type gene had no obvious effect on cell growth under normal growth conditions. The implications of these results in eIF5 function are discussed.

  • specific interaction of eukaryotic translation Initiation Factor 5 eif5 with the β subunit of eif2
    Journal of Biological Chemistry, 1997
    Co-Authors: Supratik Das, Kallol Das, Tapan Maiti, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts with the 40 S Initiation complex (40 S·mRNA· eIF3·Met-tRNAf·eIF2·GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude Initiation Factor preparations. We observed that eIF5 specifically interacted with the β subunit of Initiation Factor eIF2. No other Initiation Factors including the γ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the β subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2β deletion mutants expressed inEscherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2β. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf·eIF2·GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the β subunit of eIF2 may be critical for the hydrolysis of GTP during translation Initiation.

  • characterization of translation Initiation Factor 5 eif5 from saccharomyces cerevisiae functional homology with mammalian eif5 and the effect of depletion of eif5 on protein synthesis in vivo and in vitro
    Journal of Biological Chemistry, 1997
    Co-Authors: Tapan Maiti, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF5) interacts in vitro with the 40 S Initiation complex (40 S.AUG.Met-tRNAf.eIF2.GTP) to mediate the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. This is the first demonstration that the TIF5 gene encodes a protein involved in Initiation of translation in eukaryotic cells. Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.

  • isolation and immunochemical characterization of eukaryotic translation Initiation Factor 5 from saccharomyces cerevisiae
    Journal of Biological Chemistry, 1993
    Co-Authors: Debabrata Chakravarti, Tapan Maiti, Umadas Maitra
    Abstract:

    Eukaryotic translation Initiation Factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal Initiation complex has been purified from yeast cell lysates. The purified Factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S Initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.

Christopher J. Lucarotti - One of the best experts on this subject based on the ideXlab platform.

  • Comparative Genome Sequence Analysis of Choristoneura occidentalis Freeman and C. rosaceana
    2016
    Co-Authors: David K. Thumbi, Michel Cusson, Christopher J. Lucarotti
    Abstract:

    The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6 % and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77 % sequence identity with each other and 96.5 % and 77.8 % sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic Initiation Factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than doe

  • comparative genome sequence analysis of choristoneura occidentalis freeman and c rosaceana harris lepidoptera tortricidae alphabaculoviruses
    PLOS ONE, 2013
    Co-Authors: David K. Thumbi, Renée Lapointe, Michel Cusson, Catherine Béliveau, Christopher J. Lucarotti
    Abstract:

    The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6% and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77% sequence identity with each other and 96.5% and 77.8% sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic Initiation Factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than does ChroNPV. Our analyses suggest that it may be appropriate to consider ChocNPV and CfMNPV as variants of the same virus species.

Hiroyuki Hiraishi - One of the best experts on this subject based on the ideXlab platform.

  • Sequential eukaryotic translation Initiation Factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2β stabilizes the 48S preInitiation complex and promotes its shift to the Initiation mode.
    Molecular and cellular biology, 2012
    Co-Authors: Chingakham Ranjit Singh, Ryosuke Watanabe, Wasimul Q. Chowdhury, Hiroyuki Hiraishi, Marcelo J. Murai, Yasufumi Yamamoto, David Miles, Yuka Ikeda, Masayo Asano, Katsura Asano
    Abstract:

    During translation Initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation Initiation Factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preInitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for Initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.

  • the c terminal domain of eukaryotic Initiation Factor 5 promotes start codon recognition by its dynamic interplay with eif1 and eif2β
    Cell Reports, 2012
    Co-Authors: Rafael E Luna, Pilar Martinmarcos, Hiroyuki Hiraishi, Jagpreet S Nanda, Haribabu Arthanari, Michelle A Markus, Barak Akabayov, Alexander G Milbradt, Lunet E Luna
    Abstract:

    Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic Initiation Factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preInitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both Factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.

  • the c terminal domain of eukaryotic Initiation Factor 5 promotes start codon recognition by its dynamic interplay with eif1 and eif2β
    Elsevier, 2012
    Co-Authors: Rafael E Luna, Pilar Martinmarcos, Hiroyuki Hiraishi, Jagpreet S Nanda, Haribabu Arthanari, Michelle A Markus, Barak Akabayov, Alexander G Milbradt, Lunet E Luna
    Abstract:

    United States. Dept. of Energy. Office of Science (United States. Dept. of Energy. Office of Basic Energy Sciences. Contract DE-AC02-98CH10886)