Figure 5.

Interactions between ERdj3B and BiP are responsible for fertility at 29°C. A, GST-ERdj3B protein (WT), GST-ERdj3B(H54Q) mutant protein (H54Q), or GST alone was immobilized on glutathione Sepharose beads and incubated with total lysate prepared from Arabidopsis seedlings with (+) or without (−) 2 mm ATP. The beads were washed as described in “Materials and Methods,” and the eluate was analyzed by SDS-PAGE and immunoblotting using anti-BiP antibodies. U indicates proteins recovered in the unbound (1%) fraction; B indicates proteins recovered in the glutathione Sepharose beads (100%) fraction. B, Rhodanese was denatured at 46 μm in 6 m guanidinium-HCl for 60 min at room temperature. Unfolded rhodanese was diluted 100-fold (0.46 μm final concentration) in 50 mm Tris-HCl, pH 7.4, 1 mm CaCl₂, and 150 mm NaCl at 23°C in the absence (black line) or presence of 2.3 μm GST-ERdj3B (blue line) or GST-ERdj3B(H54Q) (red line). Aggregation (turbidity) was measured at 320 nm. C, Inflorescences of wild-type, erdj3b-1 [3b], and erdj3b-1 plants transformed with a construct expressing ERdj3B [3b(comp.)] and a construct expressing ERdj3B(H54Q) [3b(H54Q)] grown at 29°C. Note that the expression of ERdj3B(H54Q) failed to restore the temperature-sensitive low seed yield phenotype of the erdj3b-1 mutant. Seed numbers per silique are shown in Supplemental Figure S1. Bar = 10 mm.