Abstract
Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified.
Highlights
The pETM-11 vector was selected for production of the protein in E. coli ROSETTA (DE3)/pLysS, allowing the introduction of an N-terminal His-tag followed by a peptide recognized by the Tobacco Etch Virus (TEV) protease
The analysis revealed that the indicated band contained predominantly deoxyhypusine synthase, the putative first enzyme in the hypusination pathway
The recombinant protein was found to be tightly associated with deoxyhypusine synthase, the first enzyme in the hypusination pathway
Summary
They are needed to promote the synthesis of proteins containing successive residues of proline (PPP or PPG) (Gutierrez et al 2013; Ude et al 2013; Doerfel et al 2013; Schüller et al 2017). The presence of these sequences causes ribosome stalling and eIF5A/EF-P are required for resumption of elongation (Schmidt et al 2016; Melnikov et al 2016). Indispensable for this action is the characteristic and unique post-translation modification: hypusination in eIF5A and β-lysinylation in EF-P, which occurs in both proteins at a conserved lysine residue located in the N-terminal domain (Huter et al 2017)
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