Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex.
Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or deoxyhypusine synthase are essential for cell survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.
Jao
David Li-En
DL
Department of Chemistry and Chemical Biology, Rutgers-The State University of New Jersey, Piscataway, NJ 08854-8087, USA.
Chen
Kuang Yu
KY
D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002846:Chromatography, Affinity; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013058:Mass Spectrometry; D009154:Mutation; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D000097574:Eukaryotic Translation Initiation Factor 5A
D001120:Arginine; D008024:Ligands; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C063507:deoxyhypusine synthase