Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Visweswaraiah, Jyothsna; Lee, Su Jung; Hinnebusch, Alan G.; Sattlegger, Evelyn

doi:10.1074/jbc.m112.368266

Cited by 22 publications

(29 citation statements)

References 39 publications

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“…However, thus far it is unknown where on the ribosome Gcn1 binds. Supporting this idea, we also found that the Gcn − phenotype of rps10AΔ or rps10BΔ strains was exacerbated by the overexpression of eEF3, a protein shown previously to impair the formation of a functional Gcn1-ribosome complex [17]. In a yeast 2-hybrid (Y2H) screen, we found that the Gcn1 fragment encompassing amino acids 1060-1777 interacted with the small ribosomal protein 10A (Rps10A).…”

Section: Introductionsupporting

confidence: 59%

“…We found that the SM s phenotype elicited by Rps10A or Rps10B overexpression was exacerbated by the M7A mutation in Gcn1 known to specifically impair Gcn1-ribosome interaction and to reduce Gcn2 activation [3]. Studies suggest that eEF3 competes with Gcn1 for some of its ribosome-binding sites, thereby affecting Gcn1 function on the ribosome [17]. Studies suggest that eEF3 competes with Gcn1 for some of its ribosome-binding sites, thereby affecting Gcn1 function on the ribosome [17].…”

Section: Discussionmentioning

confidence: 91%

“…We have found previously that overexpression of eEF3 negatively affects Gcn2 activation and that this is due to eEF3 blocking Gcn1 function on the ribosome [17]. Studies suggested that eEF3 does not remove Gcn1 from the ribosome, but it prevents Gcn1 from accessing all its binding sites on the ribosomes, in particular those required for forming a functional Gcn1-ribosome complex.…”

Section: Rps10 Overexpression or Knockdown Exacerbates The Gcn − Phenmentioning

confidence: 99%

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Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2

Lee

Swanson

Sattlegger

2015

Biochemical Journal

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In eukaryotes, amino acid deprivation leads to the accumulation of uncharged tRNAs that are detected by Gcn2 (general control non-derepressible 2), which in turn phosphorylates eIF2α (α-subunit of eukaryotic translation initiation factor 2), an essential process for overcoming starvation. In Saccharomyces cerevisiae, sensing amino acid shortages requires that Gcn2 binds directly to its effector protein Gcn1 and both must associate with the ribosome. Our hypothesis is that uncharged tRNAs occur in the ribosomal A-site and that Gcn1 is directly involved in transfer of this starvation signal to Gcn2. In the present paper, we provide evidence that Gcn1 directly contacts the small ribosomal protein S10 (Rps10). Gcn1 residues 1060-1777 showed a yeast two-hybrid (Y2H) interaction with Rps10A. In vitro, Rps10A or Rps10B co-precipitated Gcn1[1060-1777] in an RNA-independent manner. rps10AΔ or rps10BΔ strains showed reduced eIF2α phosphorylation under replete conditions and shortly after onset of starvation, suggesting that Gcn1-mediated Gcn2 activation was impaired. Overexpression of GST-tagged Rps10 reduced growth under amino acid starvation and this was exacerbated by the Gcn1-M7A mutation known to impair Gcn1-ribosome interaction and Gcn2 activity. Under amino acid starvation, eEF3 (eukaryotic translation elongation factor 3) overexpression, known to weaken Gcn1 function on the ribosome, exacerbated the growth defect of rps10AΔ or rps10BΔ strains. Taken together, these data support the idea that Gcn1 contacts ribosome-bound Rps10 to efficiently mediate Gcn2 activation.

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Section: Introductionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 91%

Section: Rps10 Overexpression or Knockdown Exacerbates The Gcn − Phenmentioning

confidence: 99%

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Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2

Lee

Swanson

Sattlegger

2015

Biochemical Journal

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“…These findings, plus the fact that overexpression of translation elongation factor 3 impedes Gcn2 activation in vivo [29], support a model in which Gcn2 is activated by uncharged tRNA that binds first to the decoding center of a translating ribosome and is then transferred to the HisRS domain in Gcn2, and that Gcn1/Gcn20 stimulate one or both of these binding reactions involving uncharged tRNA [28].…”

Section: Introductionsupporting

confidence: 55%

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

Sebastien

Rothenburg²,

Dever³

et al. 2014

PLoS Genet

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The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn− substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd− substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd− substitutions enhance YKD-KD interactions in vitro, whereas Gcn− substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd− substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.

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“…Future work will be able to elucidate precisely the important deterministic relationship between regulatory changes in the activity of specific factors and the resulting modulation of protein synthesis in yeast cells in response to environmental stress. For example, physical and/or functional interactions between the kinase General-controlnon-derepressible-2 (Gcn2) and the elongation factors eEF1A and eEF3 seem to mediate modulation of the amino-acid starvation response in S. cerevisiae (Visweswaraiah et al, 2011(Visweswaraiah et al, , 2012, and in vivo rate control analysis could help to quantitate the regulatory impact of these interactions. The current work also represents a starting point for equivalent analyses of the distinct control properties of the protein synthesis pathways in animals and plants and how these react to stress and disease.…”

Section: Discussionmentioning

confidence: 99%

An in vivo control map for the eukaryotic mRNA translation machinery

Firczuk

Kannambath

Pahle

et al. 2013

Molecular Systems Biology

131

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Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation

Cited by 22 publications

References 39 publications

Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2

Gcn1 contacts the small ribosomal protein Rps10, which is required for full activation of the protein kinase Gcn2

Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

An in vivo control map for the eukaryotic mRNA translation machinery

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