Figure 8.

PDIL2;3 Facilitated Intermolecular Sulfhydryl Oxidation of the α-Globulin(C79F) Mutant.

(A) Proteins extracted from mature seeds of the wild type and PDIL2;3 knockdown (PDIL2;3-KD) were fractionated into the supernatant (fraction S) and pellet (fraction P) (see Methods for detail). The proteins were reduced and separated by SDS-PAGE and subjected to immunoblot analysis with anti-α-globulin (Glb) antibody.

(B) Proteins extracted from mature seeds of the wild type and the esp2 mutants expressing spGFP-PDIL1;1 or spGFP-PDIL2;3 under the control of the rice TIP3 promoter (Onda et al., 2009) were fractionated into the S and P fractions. The protein extracts were reduced and analyzed by SDS-PAGE and subjected to immunoblot analysis with anti-α-globulin antibody. Note that almost a half of α-globulin was fractionated in the P fraction when proteins were extracted from the esp2 seed (Figure 3B, lanes 3 and 4).

(C) Comparison of rRNase-refolding activities of PDIL1;1 and PDIL2;3 in vitro. The reduced, denatured RNase was refolded in the presence of recombinant PDIL1;1 or PDIL2;3 as described in Methods. The relative activities are presented as means with standard deviation of duplicate experiments.

(D) Effects of GSSG concentrations on the rate of rRNase-refolding activities of PDIL1;1 and PDIL2;3 in vitro. All assays were performed at a fixed GSH concentration of 1 mM at 28°C for 15 min. The relative activities are presented as means with standard deviation of duplicate experiments.

(E) and (F) Oxidative folding of reduced, denatured wild-type α-globulin (E) or α-globulin(C79F) mutant (F) in vitro. Protein refolding was performed in the absence (lanes 1 to 3) or presence of recombinant PDIL1;1 (lanes 4 to 6) or PDIL2;3 (lanes 7 to 9) in redox buffers containing a fixed GSH concentration of 1 mM and different GSSG concentrations of 0.05, 0.1, or 0.2 mM (see Methods for detail). The reactions were terminated at 20 min for α-globulin(WT) or 10 min for α-globulin(C79F) by adding 2× SDS sample buffer containing iodoacetamide. The reaction mixtures were centrifuged to separate the supernatant (Sup., top panels) and pellet (bottom panels). The reaction products were separated by SDS-PAGE under nonreducing [Sup. DTT(−)] or reducing condition [Pellet DTT(+)] and stained with Coomassie blue (CBB, left panels) or subjected to immunoblot analysis with anti-α-globulin antibody (right panels). Arrowheads indicate the reduced (red, apparent molecular mass of 26 kD) and oxidized (ox; apparent molecular mass of 22 kD) forms of α-globulin (Glb). The asterisk and dotted line indicate dimers and oligomers of α-globulin, respectively. Arrows indicate the bottom of sample wells.