MilliporeSigma
  • Home
  • Search Results
  • The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

PloS one (2015-07-16)
Richard C Silva, Martina Dautel, Bruno M Di Genova, David C Amberg, Beatriz A Castilho, Evelyn Sattlegger
ABSTRACT

The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

MATERIALS
Product Number
Brand
Product Description

SAFC
HEPES
Sigma-Aldrich
Anti-GAPDH antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
Sigma-Aldrich
Propidium iodide, ≥94.0% (HPLC)
Sigma-Aldrich
Sodium chloride solution, 5 M
Sigma-Aldrich
D-Sorbitol, BioXtra, ≥98%
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
D-Sorbitol, ≥98%
Sigma-Aldrich
D-Sorbitol, for molecular biology, ≥98%
Sigma-Aldrich
D-Sorbitol, BioReagent, suitable for cell culture, suitable for plant cell culture
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
HEPES, BioXtra, suitable for mouse embryo cell culture, ≥99.5% (titration)
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride, tablet
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Imidazole, ACS reagent, ≥99% (titration)
Sigma-Aldrich
Imidazole, ≥99% (titration), crystalline
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Sorbitol F solution, 70 wt. % in H2O, Contains mainly D-sorbitol with lesser amounts of other hydrogenated oligosaccharides
Sigma-Aldrich
D-Sorbitol, FCC, FG
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
D-Sorbitol, BioUltra, ≥99.0% (HPLC)
Sigma-Aldrich
Imidazole, BioUltra, ≥99.5% (GC)