Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Abstract

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

“…Formin and CAP/ Srv2p, which we have shown are depleted after eIF5A knockdown, are both involved in the regulation of actin filament assembly (47,48). Depletion of eIF5A in S. cerevisiae also resulted in misshapen cells that were more sensitive to ethanol (49). Yeast formin also contains a long poly(Pro) tract, and its translation was also adversely effected by loss of eIF5A, leading to a loss of shmoo formation (24).…”

Gender difference of androgen actions on skeletal muscle transcriptome

“…Eukaryotic translation initiation factor 5A (Eif5a) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. The depletion of Eif5a leads to a striking change of actin dynamics (disappearance of actin cables) as well as a complete cell cycle arrest at G 1 phase (Chatterjee et al 2006). Both Sat1 and Eif5a as well as Rpl15 and ubiquitinconjugating enzyme E2R 2 (Ube2r2) were modulated by DHT treatment.…”

Deoxyhypusine synthase haploinsufficiency attenuates acute cytokine signaling

“…25 Inhibition of DHS using GC7 and similar polyamines was shown to have a repressive effect on proliferation in both yeast and mammalian cell cultures as soon as 24 h, with a particularly striking inhibition at the G 1 /S transition of the cell cycle. 22,[26][27][28] The specificity of these inhibitors with respect to the hypusination pathway is at least partially verified in studies showing similar G 1 /S block in temperature-sensitive mutants of eIF5A in yeast, 18,29,30 in DOHH mutants in Drosophila, 31 and by using inhibitors of DOHH in mammalian cells. 32 Whereas all of these studies have been performed in cultured cells or in lower organisms, to date no formal accounts of eIF5A, DHS or DOHH knockouts in intact mice have been reported (of note, one review states in passing that the homozygous knockout of alleles encoding DHS is embryonic lethal).…”