Fig. 7

An eIF5A1 SUMOylation-deficient mutant cannot completely substitute for yeast HYP2. A Histidine-tagged proteins were purified from yeast cells transformed with an empty vector or with Histidine- and Flag-tagged human eIF5A1–WT under denaturing conditions. Then, purified proteins were analyzed by western blot using anti-Smt3 antibody. Arrow indicates Smt3-conjugated eIF5A1 protein. B Histidine-tagged proteins were purified from yeast cells transformed with an empty vector, Histidine- and Flag-tagged human eIF5A1–WT or Histidine- and Flag-tagged human eIF5A1–5KR under denaturing conditions. Purified proteins were then analyzed by western blot using anti-Smt3 antibody. C WT and hyp2-1 (upper row) or hyp2-3 (lower row) yeast strains were transformed with different alleles of Histidine- and Flag-tagged human eIF5A1 in a pAG425GPD vector or with the empty vector. The resulting strains were streaked on SC–Leu plates, incubated at 25 °C or 37 °C for 3 days and imaged in a GelDoc documentation system. D Expression levels of the different versions of eIF5A in the indicated strains, growing at 25 °C or after 4 h at 37 °C, were analyzed by western blot. Ponceau staining of the membrane is shown as a loading control