Fig. 8

SUMOylation of eIF5A1 is important for polysome disassembly and SG formation upon heat-shock stress. A Yeast cells with temperature-sensitive eIF5A mutant hyp2-1 were transformed with different alleles of Histidine- and Flag-tagged human eIF5A1 in a pAG425GPD vector or with the empty vector. Cells were grown in SC–Leu medium until early exponential phase, incubated at 37 °C for 4 h to deplete endogenous eIF5A and then subjected to severe heat shock stress (46 °C, 30 min). Representative polysome profiles after gradient fractionation of yeast extracts are shown and the ribosomal subunits (40S and 60S), monosomes (80S), and polysomes are indicated (upper panel). When translation is arrested during stress, a reduction in the intensity of polysome peaks is seen because fewer ribosomes initiate translation. At the same time, the 60S and 80S peaks increase. The percentage of polysomes from three (eIF5A WT, vector, eIF5A–K50R, eIF5A–3KA) or two (eIF5A–3KR) independent experiments is shown as the median and standard deviation (lower panel). Statistical analysis was assessed by a Student’s t-test. *P < 0.05. B hyp2-1 yeast strains were cotransformed with the SG marker Pab1–GFP together with the Histidine- and Flag-tagged human eIF5A1 WT or 5KR or an empty vector. WT yeast transformed with Pab1–GFP was included as a positive control. The resulting strains were incubated at 25 °C or heat shocked at 46 °C for 10 min and the formation of SG was then evaluated using a fluorescence microscope