title: Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.
abstract: Cells respond to changes in their environment through mechanisms that often necessitate reprogramming of the translation machinery. The fastest and strongest of all tested responses is the translation inhibition observed following abrupt depletion of glucose from the media of yeast cells. The speed of the response suggests a post-translational modification of a key component of the translation machinery. This translation factor is as yet unknown. A cAMP-dependent protein kinase mutant yeast strain (tpk1(w)) that does not respond properly to glucose depletion and maintains translation was described previously. We hypothesized that the inability of tpk1(w) to arrest translation results from abnormal expression of key translation mediators. Genome-wide analysis of steady-state mRNA levels in tpk1(w) revealed underexpression of several candidates. Elevating the cellular levels of eukaryotic initiation factor (eIF) 5 by overexpression rescued the translational defect of tpk1(w). Restoring ribosomal dissociation by eIF5 necessitated an active GAP domain and multiple regions throughout this protein. Phosphoproteomics analysis of wild-type cells overexpressing eIF5 revealed increased phosphorylation in a novel site (Thr191) upon glucose depletion. Mutating this residue and introducing it into tpk1(w) abolished the ability of eIF5 to rescue the translational defect. Intriguingly, introducing this mutation into the wild-type strain did not hamper its translational response. We further show that Thr191 is phosphorylated in vitro by Casein Kinase II (CKII), and yeast cells with a mutated CKII have a reduced response to glucose depletion. These results implicate phosphorylation of eIF5 at Thr191 by CKII as one of the pathways for regulating translation upon glucose depletion.
authors.affiliation: Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel.
authors.forename: Ira
authors.initials: I
authors.lastname: Bavli-Kertselli
authors.forename: Daniel
authors.initials: D
authors.lastname: Melamed
authors.forename: Lavi
authors.initials: L
authors.lastname: Bar-Ziv
authors.forename: Hila
authors.initials: H
authors.lastname: Volf
authors.forename: Yoav
authors.initials: Y
authors.lastname: Arava
mesh_terms: D040061:Eukaryotic Initiation Factor-5 / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D040061:Eukaryotic Initiation Factor-5; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins
keywords: environmental changes; eukaryotic initiation factor 5 (eIF5); glucose depletion; phosphorylation; polysomes