Revised Manuscript Received July 31, 1995® abstract: 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-5Afrom 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 has 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/3-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to themain form, all three minor isoforms of eIF-5A are characterized by acetylation of the e-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 themain 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.

Only one protein is known to contain the unusual amino acid hypusine: eukaryotic initiation factor 5 A (elF-SA). 1In this protein, a specific lysine residueis modified via transfer and subsequent hydroxylation of an aminobutyl moiety from spermidine to its e-amino group (Wolff et al., 1990). elF-5A with its hypusine modification is highly conserved in all