Figure 3

Manganese rescue of Ire1 RNase activity and oligomerization in the presence of ADPβS. (a) Structures of ADP and ADPβS. (b) and (c) The effect of increasing Mg²⁺ and Mn²⁺ concentrations on Ire1 RNase activity in the presence of 2 mmol ADPβS (b) or 2 mmol ADP (c). The buffer contained 0.2 mmol Mg²⁺. All reactions contained 3 μmol Ire1KR32. Assays were conducted as described in Figure 1(a)a. (d) Visible oligomerization of Ire1KR32 in the absence of cofactors and in the presence of 1 mmol ADP, 1 mmol ADPβS, 1 mmol Mn²⁺ and 1 mmol ADPβS + 1 mmol Mn²⁺. Assays contained 10 μmol Ire1KR32 and were conducted as described in Figure 1(b). All reactions contained 2 mmol Mg²⁺. The bar chart below the image shows relative opacity of each sample. (e) Cooperative activation of Ire1KR32 RNase assayed using ³²P-5'-HP21 substrate as described in Figure 1(a). Reactions contained 2 mmol ADPβS, 2 mmol Mg²⁺ and either 0 or 2 mmol Mn²⁺ as indicated. (f) OD₅₀₀ versus concentration of Ire1KR32 in the presence of 2 mmol ADP, 2 mmol ADPβS and 2 mmol ADPβS + 2 mmol Mn²⁺. All reactions contained 2 mmol Mg²⁺. (g) Interactions involving the β-phosphate in the Ire1 ADP Mg complex (PDB ID 2rio). Note that all three nonbridging oxygen atoms of the β-phosphate form contacts to either protein residues or Mg²⁺. (h) The rate constants for Ire1 and Ire1(D828A) in the presence of ADP Mg²⁺ and APY29. Error bars show standard errors of single-exponential fitting of corresponding time courses.