scholarly journals Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1920-1932 ◽  
Author(s):  
J L Bushman ◽  
A I Asuru ◽  
R L Matts ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Open Reading Frames ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Molecular Weight Complex ◽  
Exchange Factor

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.

Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (3) ◽  
pp. 1920-1932
Author(s):  
J L Bushman ◽  
A I Asuru ◽  
R L Matts ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Open Reading Frames ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Molecular Weight Complex ◽  
Exchange Factor

Starvation of the yeast Saccharomyces cerevisiae for an amino acid signals increased translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. We have isolated and characterized the GCD6 and GCD7 genes and shown that their products are required to repress GCN4 translation under nonstarvation conditions. We find that both GCD6 and GCD7 show sequence similarities to components of a high-molecular-weight complex (the GCD complex) that appears to be the yeast equivalent of translation initiation factor 2B (eIF-2B), which catalyzes GDP-GTP exchange on eIF-2. Furthermore, we show that GCD6 is 30% identical to the largest subunit of eIF-2B isolated from rabbit reticulocytes. Deletion of either GCD6 or GCD7 is lethal, and nonlethal mutations in these genes increase GCN4 translation in the same fashion described for defects in known subunits of eIF-2 or the GCD complex; derepression of GCN4 is dependent on short open reading frames in the GCN4 mRNA leader and occurs independently of eIF-2 alpha phosphorylation by protein kinase GCN2, which is normally required to stimulate GCN4 translation. Together, our results provide evidence that GCD6 and GCD7 are subunits of eIF-2B in S. cerevisiae and further implicate this GDP-GTP exchange factor in gene-specific translational control.

Identification of GCD14 and GCD15, Novel Genes Required for Translational Repression of GCN4 mRNA in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1007-1020 ◽  
Author(s):  
Rafael Cuesta ◽  
Alan G Hinnebusch ◽  
Mercedes Tamame
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
New Genes ◽  
General Translation

Abstract In Saccharomyces cerevisiae, expression of the transcriptional activator GCN4 increases at the translational level in response to starvation for an amino acid. The products of multiple GCD genes are required for efficient repression of GCN4 mRNA translation under nonstarvation conditions. The majority of the known GCD genes encode subunits of the general translation initiation factor eIF-2 or eIF-2B. To identify additional initiation factors in yeast, we characterized 65 spontaneously arising Gcd− mutants. In addition to the mutations that were complemented by known GCD genes or by GCN3, we isolated mutant alleles of two new genes named GCD14 and GCD15. Recessive mutations in these two genes led to highly unregulated GCN4 expression and to derepressed transcription of genes in the histidine biosynthetic pathway under GCN4 control. The derepression of GCN4 expression in gcd14 and gcd15 mutants occurred with little or no increase in GCN4 mRNA levels, and it was dependent on upstream open reading frames (uORFs) in GCN4 mRNA that regulate its translation. We conclude that GCD14 and GCD15 are required for repression of GCN4 mRNA translation by the uORFs under conditions of amino acid sufficiency. The gcd14 and gcd15 mutations confer a slow-growth phenotype on nutrient-rich medium, and gcd15 mutations are lethal when combined with a mutation in gcd13. Like other known GCD genes, GCD14 and GCD15 are therefore probably required for general translation initiation in addition to their roles in GCN4-specific translational control.

scholarly journals A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

2021 ◽  
Author(s):  
Lan Wang ◽  
Morgane Boone ◽  
Rosalie E Lawrence ◽  
Adam Frost ◽  
Peter Walter ◽  
...  
Keyword(s):  
Stress Response ◽  
Point Mutation ◽  
Translational Control ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Nucleotide Exchange Factor

AbstractIn eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.

The Gcn2–eIF2α pathway connects iron and amino acid homeostasis in Saccharomyces cerevisiae

2018 ◽  
Vol 475 (8) ◽  
pp. 1523-1534 ◽  
Author(s):  
Marcos Caballero-Molada ◽  
María D. Planes ◽  
Helena Benlloch ◽  
Sergio Atares ◽  
Miguel A. Naranjo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Iron Content ◽  
Amino Acid Biosynthesis ◽  
Initiation Factor ◽  
Acid Biosynthesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Iron Sulfur ◽  
Amino Acid Homeostasis

In eukaryotic cells, amino acid biosynthesis is feedback-inhibited by amino acids through inhibition of the conserved protein kinase Gcn2. This decreases phosphorylation of initiation factor eIF2α, resulting in general activation of translation but inhibition of translation of mRNA for transcription factor (TF) Gcn4 in yeast or ATF4 in mammals. These TFs are positive regulators of amino acid biosynthetic genes. As several enzymes of amino acid biosynthesis contain iron–sulfur clusters (ISCs) and iron excess is toxic, iron and amino acid homeostasis should be co-ordinated. Working with the yeast Saccharomyces cerevisiae, we found that amino acid supplementation down-regulates expression of genes for iron uptake and decreases intracellular iron content. This cross-regulation requires Aft1, the major TF activated by iron scarcity, as well as Gcn2 and phosphorylatable eIF2α but not Gcn4. A mutant with constitutive activity of Gcn2 (GCN2c) shows less repression of iron transport genes by amino acids and increased nuclear localization of Aft1 in an iron-poor medium, and increases iron content in this medium. As Aft1 is activated by depletion of mitochondrial ISCs, it is plausible that the Gcn2–eIF2α pathway inhibits the formation of these complexes. Accordingly, the GCN2c mutant has strongly reduced activity of succinate dehydrogenase, an iron–sulfur mitochondrial enzyme, and is unable to grow in media with very low iron or with galactose instead of glucose, conditions where formation of ISCs is specially needed. This mechanism adjusts the uptake of iron to the needs of amino acid biosynthesis and expands the list of Gcn4-independent activities of the Gcn2–eIF2α regulatory system.

GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 506-520
Author(s):  
E M Hannig ◽  
A M Cigan ◽  
B A Freeman ◽  
T G Kinzy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Metal Binding ◽  
Critical Role ◽  
Cross Reactivity ◽  
Translation Initiation Factor ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Gamma Subunit

The eukaryotic translation initiation factor eIF-2 plays a critical role in regulating the expression of the yeast transcriptional activator GCN4. Mutations in genes encoding the alpha and beta subunits of eIF-2 alter translational efficiency at the GCN4 AUG codon and constitutively elevate GCN4 translation. Mutations in the yeast GCD11 gene have been shown to confer a similar phenotype. The nucleotide sequence of the cloned GCD11 gene predicts a 527-amino-acid polypeptide that is similar to the prokaryotic translation elongation factor EF-Tu. Relative to EF-Tu, the deduced GCD11 amino acid sequence contains a 90-amino-acid N-terminal extension and an internal cysteine-rich sequence that contains a potential metal-binding finger motif. We have identified the GCD11 gene product as the gamma subunit of eIF-2 by the following criteria: (i) sequence identities with mammalian eIF-2 gamma peptides; (ii) increased eIF-2 activity in extracts prepared from cells cooverexpressing GCD11, eIF-2 alpha, and eIF-2 beta; and (iii) cross-reactivity of antibodies directed against the GCD11 protein with the 58-kDa polypeptide present in purified yeast eIF-2. The predicted GCD11 polypeptide contains all of the consensus elements known to be required for guanine nucleotide binding, suggesting that, in Saccharomyces cerevisiae, the gamma subunit of eIF-2 is responsible for GDP-GTP binding.

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae

1991 ◽  
Vol 11 (6) ◽  
pp. 3105-3114
Author(s):  
J Schnier ◽  
H G Schwelberger ◽  
Z Smit-McBride ◽  
H A Kang ◽  
J W Hershey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Peptide Bond ◽  
Translation Initiation Factor ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Mutant Form ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

scholarly journals Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Science ◽  
2018 ◽  
Vol 359 (6383) ◽  
pp. eaaq0939 ◽  
Author(s):  
Jordan C. Tsai ◽  
Lakshmi E. Miller-Vedam ◽  
Aditya A. Anand ◽  
Priyadarshini Jaishankar ◽  
Henry C. Nguyen ◽  
...  
Keyword(s):  
Competitive Inhibitor ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Cryo Electron Microscopy ◽  
Nucleotide Exchange Factor ◽  
Initiation Factor Eif2 ◽  
Resolution Structure

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo–electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

Invited Review: Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise

2002 ◽  
Vol 93 (3) ◽  
pp. 1168-1180 ◽  
Author(s):  
Scot R. Kimball ◽  
Peter A. Farrell ◽  
Leonard S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Initiation Factor ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Amino Acid Availability

Protein synthesis in skeletal muscle is modulated in response to a variety of stimuli. Two stimuli receiving a great deal of recent attention are increased amino acid availability and exercise. Both of these effectors stimulate protein synthesis in part through activation of translation initiation. However, the full response of translation initiation and protein synthesis to either effector is not observed in the absence of a minimal concentration of insulin. The combination of insulin and either increased amino acid availability or endurance exercise stimulates translation initiation and protein synthesis in part through activation of the ribosomal protein S6 protein kinase S6K1 as well as through enhanced association of eukaryotic initiation factor eIF4G with eIF4E, an event that promotes binding of mRNA to the ribosome. In contrast, insulin in combination with resistance exercise stimulates translation initiation and protein synthesis through enhanced activity of a guanine nucleotide exchange protein referred to as eIF2B. In both cases, the amount of insulin required for the effects is low, and a concentration of the hormone that approximates that observed in fasting animals is sufficient for maximal stimulation. This review summarizes the results of a number of recent studies that have helped to establish our present understanding of the interactions of insulin, amino acids, and exercise in the regulation of protein synthesis in skeletal muscle.

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