A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (research article)

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dc.citation.issue11
dc.citation.volume17
dc.contributor.authorGottfried S
dc.contributor.authorKoloamatangi SMBMJ
dc.contributor.authorDaube C
dc.contributor.authorSchiemann AH
dc.contributor.authorSattlegger E
dc.contributor.editorLustig AJ
dc.coverage.spatialUnited States
dc.date.accessioned2024-01-10T01:27:31Z
dc.date.accessioned2024-07-25T06:47:42Z
dc.date.available2022-11-28
dc.date.available2024-01-10T01:27:31Z
dc.date.available2024-07-25T06:47:42Z
dc.date.issued2022-11-28
dc.description.abstractThe protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.
dc.description.confidentialfalse
dc.format.paginatione0277648-
dc.identifier.author-urlhttps://www.ncbi.nlm.nih.gov/pubmed/36441697
dc.identifier.citationGottfried S, Koloamatangi SMBMJ, Daube C, Schiemann AH, Sattlegger E. (2022). A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation.. PLoS One. 17. 11. (pp. e0277648-).
dc.identifier.doi10.1371/journal.pone.0277648
dc.identifier.eissn1932-6203
dc.identifier.elements-typejournal-article
dc.identifier.issn1932-6203
dc.identifier.numbere0277648
dc.identifier.piiPONE-D-22-05688
dc.identifier.urihttps://mro.massey.ac.nz/handle/10179/70889
dc.languageeng
dc.publisherPLOS
dc.publisher.urihttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0277648
dc.relation.isPartOfPLoS One
dc.rights(c) 2022 The Author/s
dc.rightsCC BY 4.0
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectAmino Acid Substitution
dc.subjectAmino Acids
dc.subjectEukaryotic Initiation Factor-2
dc.subjectPeptide Elongation Factors
dc.subjectProtein Serine-Threonine Kinases
dc.subjectRNA-Binding Proteins
dc.subjectSaccharomyces cerevisiae
dc.subjectSaccharomyces cerevisiae Proteins
dc.subjectTrans-Activators
dc.titleA genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (research article)
dc.typeJournal article
pubs.elements-id458273
pubs.organisational-groupOther
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