Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression.
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.
Chatterjee
Ishita
I
Department of Chemistry and Chemical Biology, Rutgers-The State University of New Jersey, Piscataway, NJ 08854-8087, USA.
Gross
Stephane R
SR
Kinzy
Terri Goss
TG
Chen
Kuang Yu
KY
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D005434:Flow Cytometry; D005800:Genes, Fungal; D016296:Mutagenesis; D009154:Mutation; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D000097574:Eukaryotic Translation Initiation Factor 5A
D000199:Actins; D017931:DNA Primers; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A