Figure 1.

tOE17-20F3C precursors form disulphide-linked dimers upon membrane binding that are subsequently transported to the thylakoid lumen. (A) In vitro translated [³H] tOE17-20F3C precursor containing a unique cysteine at residue 3 of the mature domain (tp) was incubated with washed thylakoids in a binding reaction. The assay mixture was separated by centrifugation into supernatant (lane S) and membranes, which were washed with IB (lane B). An aliquot of precursor-bound thylakoids was further incubated in a chase assay (lane C) in the absence (lanes 4 and 8) or presence (lanes 5 and 9) of nigericin and valinomycin (nig/val), or presence (lanes 6 and 10) of DTT as depicted below the panel. Aliquots of the thylakoid fraction were treated with the protease thermolysin (lanes 7–10). Samples were analysed by SDS–PAGE without reducing agent and fluorography. Putative precursor dimers and processed lumenal mature protein dimers (PD), monomer precursor and processed lumenal monomer (PM) are indicated to the right of the panel. (B) The same samples as in (A) were treated with β-mercaptoethanol before SDS–PAGE and fluorography. (C) Binding reactions were conducted with full length tOE17-20F3C and/or tOE17-20F3C truncated after residue 100 of the mature domain (labelled tOE17-20F3CTr). Lanes 1 and 2 contain the individual in vitro translation products of tOE17-20F3C and tOE17-20F3CTr. Lanes 3, 4 and 5 are recovered thylakoids membrane from binding assays with tOE17-20F3C, tOE17-20F3CTr and a mixture of the two precursors, respectively. tOE17-20F3C monomers and dimers (F monomer and F Dimer), tOE17-20F3CTr monomers and dimer (Tr monomer and Tr dimer) and the tOE17-20F3C:tOE17-20F3CTr heterodimer (F+Tr Dimer) are indicated to the right of the panel. Mrs in parentheses (in kilodaltons) of the different protein species were determined from their relative mobility fit to a curve of pre-stained molecular mass standards (Fisher Chemical Co.). (D) In vitro translated [³H] tOE17-20F3C precursor (lane 1) was incubated with washed thylakoids in a transport assay in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of nigericin and valinomycin. Aliquots of recovered thylakoids were treated with thermolysin as designated below the panel. Samples were analysed by non-reducing SDS–PAGE and fluorography.