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.