Archaea / 2016 / Article / Fig 3

Research Article

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

Figure 3

aIF5A produced in Hfx. volcanii is deoxyhypusinylated and sensitive to oxidative stress. (a) Hfx. volcanii H26 was grown at 42°C in ATCC974 (shaking at 200 rpm). 2 mL of samples at OD600 = 0.043 was harvested along the growth curve. (b) H26 cells pellets (2 mL at OD600 = 0.043 of the culture) were resuspended in SDS-PAGE loading buffer and boiled for 15 min. Equivalent protein loading was based on OD600 of cell culture (0.086 OD600 units per lane), as demonstrated by staining with Coomassie Brilliant Blue R-250 (CB stain, with representative 55 kDa protein band of Hfx. volcanii presented). Proteins were separated via 4–15% reducing SDS-PAGE. aIF5A was detected by α-aIF5A (anti-TIF5A2) immunoblot (IB). The experiments were performed with three biological replicates and the detection by anti-TIF5A2 or anti-TIF5A3. The molecular mass indicated is in kDa. (c) Effect of oxidative stress on aIF5A level. The cells were grown for 24 hours and then treated or not treated with 0.78% H2O2. Equivalent protein loading was based on OD600 of cell culture (0.086 OD600 units per lane). Lane 1, cells collected at 24 hours; lane 2, cells collected at +4 hours after no stress (control experiment); lane 3, cells collected at +4 hours after 0.78% H2O2 treatment. Proteins were separated by 4–15% reducing SDS-PAGE. aIF5A was detected via α-aIF5A (anti-TIF5A2) immunoblot (IB). The molecular mass indicated is in kDa. (d) Identification of the deoxyhypusine modification by LC-MS/MS analysis. The purified aIF5A His-C-term was loaded on a SDS-PAGE 12%. The proteins were detected by staining with Coomassie Blue (Fig S2B), and the protein band was cut and analyzed by LC-MS/MS analysis. Fragmentation spectrum for deoxyhypusinylated peptide PGKHGSAK is shown; xiSPEC (http://spectrumviewer.org/) was used for annotation.
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