Posttranslational modification of a specific lysine residue in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability and proliferation. The product of this modification is hypusine, an amino acid unique to eIF-5A. We have purified and characterized one major and three minor isoforms of human eIF-5A from HeLa cells. The main form, which accounts for approximately 95% of the total eIF-5A, carries hypusine at position 50 and is amino-terminally acetylated as determined by amino acid composition analysis and electrospray ionization mass spectrometry. Analytical gel filtration indicates that this protein variant possesses a native apparent molecular weight that lies between that expected for a monomeric and dimeric form. Nevertheless, several experiments confirm this protein to be monomeric. It is further shown that eIF-5A have well-defined secondary structure. Both the far-UV circular dichroism spectrum as well as secondary structure predictions using different algorithms suggest this protein to have predominantly beta-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to the main form, all three minor isoforms of eIF-5A are characterized by acetylation of the epsilon-amino group of lysine at position 47. The minor isoforms are distinguishable by their state of modification of the lysine residue at position 50. Whereas the main form occurs in both the cytoplasmic and the nuclear fraction of HeLa cells, the minor isoforms were not detectable in the preparation of the nuclear fraction. Therefore, acetylation of lysine at position 47 might play a controlling role in the distribution of the minor isoforms to the nucleus.
In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.
The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.
The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals.
Electrospray mass s~trometry of the purified isoforms of the hypusine-containing protein of ~ucc~ro~yces cerevisiae HypZp suggested a phospho~lation of the acidic isoform, which was confirmed by phosphatase treatment. The phospho~lation site was mapped to the N-acetylated se&e residue in position no. 1 by mass spectrometric analysis of enzymatic fragments. Mutation of this serine residue gives rise to only the basic isoform, confirming our protein chemical data. As this mutation has no effect on cell viability or growth rate. the unphosphorylated isoform is sufficient to exert the essential in vivo function of Hyp2p.
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