Modification of translation factor aIF5A from Sulfolobus solfataricus

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.Electronic supplementary materialThe online version of this article (10.1007/s00792-018-1037-4) contains supplementary material, which is available to authorized users.

Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus

“…In addition to a~55 kDa band, which was previously identified as the aCPSF2 5´to 3é xoribonuclease [17,28], a band was visible at a position, which corresponded to 15 kDa, approximating to the size of aIF5A (Figure 3(a)). Hypusinated aIF5A was then enriched from Sso P2 [6] (Figure 3(b)). To test further whether the observed RNase activity (Figure 3(a)) originated from Sso aIF5A the zymogram assay described above was exploited again.…”

“…Nevertheless, the mass spectrometry analysis also identified several other proteins (Table S1, Figure S2). Therefore, we also purified a hypusinated C-terminally Histagged variant of aIF5A [6] from Sso strain PH1-16(pMJ05-aIF 5A-C-His). As shown in Figure 3 when the RNA was incubated with a concentrated eluate obtained after mock purification using a lysate of the control strain Sso PH1-16(pMJ05-ptf55α), suggesting that aIF5A is indeed endowed with RNase activity.…”

“…Next, we asked whether unmodified Sso N-His-aIF5A [6] purified to near homogeneity from E. coli (Figure 4(a)) is able to degrade 2508sh RNA. As shown in Figure 4(b), the degradation of the RNA substrate occurred at 65°C either in presence (Panel I) or absence of Mg ++ (Panel II).…”

“…In contrast, degradation of 2508sh RNA did not occur at 37°C (Figure 4 (b), Panel III) suggesting that the aIF5A ribonucleolytic activity is only exerted under thermophilic conditions. To further verify that the ribonucleolytic activity of N-His-aIF5A preparation purified from E. coli is intrinsic to N-His-aIF5A, 2508sh RNA was incubated with a His-tagged version of recombinant Sso deoxyhypusine synthase (DHS), which was purified from E. coli using the same procedure as for N-His-aIF5A [6]. As shown in Figure 4(b), panel IV, no ribonucleolytic activity was observed, revealing that the ribonucleolytic activity of N-His-aIF5A derived from E. coli is not attributable to contaminant ribonucleases.…”

“…The eukaryal protein is hypusinated [4], whereas the bacterial orthologue is βlysinylated [5]. It was further shown that the archaeal protein is either hypusinated or deoxyhypusinated, and that some Archaea contain both versions of the protein [6,7]. The 3D structures of eIF5A and aIF5A are very similar, with a strong conservation of residues in the N-terminal moiety that contains the eIF5A hypusination site [8].…”