Fig. 4.

Thioredoxin-dependent post-translational rearrangement of ER-localised nascent chain disulfides. (A) Cartoon depicting the rearrangement of nascent chain disulfides in a stalled translocation intermediate following the addition of a cytosol-localised reduction system. (B) Schematic of the disulfide pattern within the disintegrin domain of ADAM10. The stalled intermediate used in subsequent experiments has the indicated disulfide-forming cysteines in the ER lumen. (C) Translations carried out in the presence of DTT (5 mM) gave rise to distinct translation products that migrate with the sizes of the pre-protein and mature protein as indicated (lane 1). Translations were carried out in the absence of added G6P and SP cells subsequently incubated post-translationally in a reticulocyte lysate (RRL) in the absence (lane 2) or presence (lane 3) of G6P. SP cells were isolated from the translations and resuspended in KHM buffer alone (−C) (lane 4). Note the rearrangement of nascent chain disulfides when the TrxR1 system is included in the incubation (lane 5) (+C) and the dependence of this rearrangement on the presence of Trx1 (lane 6) (C-T). The experiment was repeated once (n=2). (D) Similarly to the TrxR1 system, TCEP brings about the rearrangement of nascent chain disulfides when added to SP cells post-translationally either in the presence of RRL (lane 3) or in KHM buffer alone (lane 5). The experiment was repeated once (n=2).