Figure 1.
Figure 1. Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.

Contact between the Tha4 TM and cpTatC TM4 in the translocase. (A) Radiolabeled pre-cpTatC L231C was imported into chloroplasts. Recovered thylakoids were incubated with in vitro–translated unlabeled Tha4 single Cys variants (XnC) followed by in vitro–translated SpF16 Tat substrate (see Materials and methods). Samples at 15°C were illuminated to assemble the translocase and CuP was added to promote disulfide formation (Materials and methods). Analysis was by SDS-PAGE/fluorography under nonreducing or reducing (+ β-mercaptoethanol) conditions. (B) Assays received SpF16 or mock translation extract before cross-linking. (C) Assays received either the full-size substrate tOE17-20F or the inactive twin lysine variant (KK-tOE17-20F). (D) Models of Tha4 and cpTatC with the positions of Cys substitutions marked with stars. Tha4 F4C E10Q is a nonfunctional Tha4 variant.

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