Table 1.
Yeast strains used in this study.
Figure 1.
Sequence alignment ofS. pombe Iph1 and hIDE.
Identities are box-shaded in blue and similarities in grey. Non-conserved Cys residues are shaded in yellow and conserved Cys residues in magenta. Residues important for catalysis are box-shaded as follows: residues required for Zn2+ coordination, cyan; Glu residue involved in catalysis, red (E71 in Iph1 and E111 in hIDE); E71/E111 were substituted by Asp in strains iph1-E71D and hIDE-E111D. Residues important for substrate recognition and fixation are shaded in green and those required for interaction between the catalytic and the substrate binding domains are shaded in black [48].
Table 2.
Sequence comparisons of hIDE and some peptidases of the M16 family.
Figure 2.
Structures of hIDE and model of Iph1 in complex with the insulin B chain.
A) Crystallographic structure of the human IDE-E111Q–insulin B chain complex (PDB code 2G56) [48]. Domains 1, 2, 3 and 4 are colored green, blue, yellow and red, respectively. The Zn2+ ion and insulin B chain are colored magenta and orange, respectively. B) Model of an insulin B chain/Iph1 complex. The four domains of Iph1 are drawn in the same orientation and color codes as in A. The two Cys conserved in hIDE are in yellow, the remaining Cys residues are in cyan.
Figure 3.
Iph1 and hIDE have similar protease activity.
A) SDS-PAGE of Iph1 elution fractions at 300 mM imidazole after Ni-NTA purification. 1: Iph1 wt, 2: Iph1-E71D. B) Activity of Iph1 WT, Iph1-E71D and hIDE towards Mca-RPPGFSAFK(Dnp). Error bars represent the standard deviation of 3 experiments. C) Titration of 1,10-phenanthroline inhibition of Mca-RPPGFSAFK(Dnp) digestion by Iph1 or hIDE. Activity is expressed as the percentage of activity of the enzyme in the absence of inhibitor. The error bars represent the standard deviation of duplicates. D) Reversed phase HPLC analysis of insulin digestions. Insulin was digested for 16 h at 37°C in the presence of 500 ng hIDE or 800 ng Iph1. E) Tricine SDS-PAGE analysis of insulin digestions. 185 ng insulin were digested for 16 h with Iph1 wt, hIDE or Iph1-E71D (0.7 or 3.5 or 14 ng). The left hand lanes show different dilutions of undigested recombinant insulin, corresponding to 100%, 50% and 25% of the starting amount used in digestions. One out of 3 experiments performed is shown. F) The percentage of digestion by the different enzymes was calculated by measuring insulin band intensity on Tricine SDS-PAGE gels.
Figure 4.
Lack of Iph1 protects cells from ER stress.
A) Drop test of wt and iph1-d strains grown in the indicated medium and exposed or not to TU for 45 min. B) Survival of the indicated strains to TU. The mean (+/− S.E.M) of seven independent experiments is shown. Mann Whitney test was used for statistical analysis. C) Survival to 1 h treatment with different concentrations of TU of wt, iph1-d and protease mutant iph1-E71D. For all strains the mean (+/− S.E.M.) of three independent experiments is shown. ANOVA with Tukey's test was applied for statistical analysis.
Figure 5.
Iph1 loss suppresses the sensitivity to proteotoxic stress oftor1 mutant in a TORC1-dependent manner.
A) Survival to TU (left panel) and DTT (right panel) of the indicated strains. For all strains the mean (+/− S.E.M.) of four independent experiments is shown. B) Survival to RA or TU and RA of the indicated strains. For all strains the mean (+/− S.E.M.) of three independent experiments is shown. C) Cell extracts from tor2-51 and iph1-d tor2-51 cells grown at 25°C and shifted to 36°C for 45 min were probed with anti-phospho-AKT Substrate antibodies recognizing the phosphorylated form of p27. Sty1 was detected as loading control, using anti-Hog1 antibodies. D) Survival to TU of the indicated strains. The mean (+/− S.E.M.) of four independent experiments is shown. For all experiments ANOVA with Tukey's test was applied for statistical analysis.
Figure 6.
Iph1 has a pro-apoptotic function during ER-stress.
A) Model resuming the principal findings in this study. See text for explanation. B) Cell extracts from wt and iph1-d cells exposed to TU for the indicated times and cell extracts from wt cells exposed or not to RA were probed with antibodies recognizing the Tor2-dependent phosphorylated form of p27. Sty1 was detected as loading control, using anti-Hog1 antibodies. C) wt and iph1-d cells were treated or not with TU for 45 min and released in fresh medium for 0, 4 and 6 hours. At each time point the metacaspase activity was detected with the fluorescent probe FITC-VAD-FMK. Bars are the mean of three independent experiments. At 6 hours the percentage of metacaspase positive test is statistically different with a p value of 0.0046.
Figure 7.
hIDE and hIDE-E111D complementiph1-d cells when expressed at the genomic locus.
A) Cell extracts from two independent clones (1 and 2) expressing hIDE or hIDE-E111D at the iph1 genomic locus were probed with anti-IDE antibodies. Wt indicates protein extract from a strain not expressing the human protein. rec hIDE indicates purified recombinant hIDE carrying a poly-His extension with an expected MW of 114 kDa. The star indicates a possible degradation product. B) Survival to 1 h treatment with different concentrations of TU of the indicated strains. The mean (+/− S.E.M.) of three independent experiments is shown and ANOVA with Tukey's test was applied for statistical analysis.