Figure 8.

Evidence for the interaction of TRX m1, TRX m2, and TRX m4 proteins with PSII protein complexes. A, Analysis of thylakoid protein complexes by BN-PAGE. Freshly isolated thylakoid membranes from protoplasts transformed with pUC19-TRX m1^C107S-FLAG, pUC19-TRX m2^C113S-FLAG, pUC19-TRX m4^C119S-FLAG, and pUC19-TRX m3^C100S-FLAG plasmids were solubilized with 1% (w/v) DM at a chlorophyll concentration of 15 μg, and the protein samples were separated by BN-PAGE. Protoplasts transformed with the empty transient expression vector were the negative control (CK). Assignments of the thylakoid membrane macromolecular protein complexes indicated at right were identified according to a previous study (Peng et al., 2006). B, Two-dimensional BN/SDS-PAGE fractionation and immunoblot analysis of PSII protein complexes. Thylakoid membrane complexes separated in A were further subjected to second-dimension SDS-urea-PAGE, and the proteins were immunodetected with specific antibodies against FLAG-tagged TRX m proteins, D1, D2, CP43, and CP47. The asterisks indicate nonspecific signals on the immunoblots. The arrows indicate the FLAG-tagged TRX m proteins. Two biological repeats were conducted, and similar results were obtained. C, Pull-down assay. His-TRX m1^C107S, His-TRX m2^C113S, His-TRX m3^C100S, and His-TRX m4^C119S recombinant proteins were immobilized on cyanogen bromide-activated Sepharose 4B resin and incubated with total thylakoidal membrane proteins solubilized with 1% (w/v) DM; empty cyanogen bromide-activated Sepharose 4B resin with no coupled His-TRX m protein was used as the negative control. After incubating and washing, proteins interacting with the His-TRX m mutant proteins were eluted with 10 mm DTT and detected by immunoblot analysis. Identities of thylakoid membrane protein complexes and their subunits are indicated at right. Three biological repeats were performed, and similar results were obtained. [See online article for color version of this figure.]