Tnt coupled reticulocyte system

[³⁵S]-methionine radiolabelled proteins were translated using the TNT^® Coupled Reticulocyte system (Promega) according to manufacturer’s instructions. Protein uptake assays were carried out into either freshly isolated mitochondria or freshly isolated plastids as previously described (Duncan et al., 2015 (link)). To confirm the intra-mitochondrial localization of imported protein, the outer membrane was ruptured by osmotic shock following import and prior to the addition of Proteinase K (PK) as detailed previously (Duncan et al., 2015 (link)). Plastid protein uptake assays were carried out by incubating precursor protein with isolated plastids (30 ug) in 3 mM ATP, 10 mM Met, 10 mM Cys, 20 mM potassium gluconate, 10 mM NaHCO₃, 3 mM MgSO₄, 330 mM sorbitol, 50 mm HEPES-KOH, pH 8, 0.2% BSA, and 50 mM ascorbic acid) for 25°C for 5 min with gentle agitation. Plastids were and washed in cold 50 mM HEPES-KOH, pH 8, 0.3 m sorbitol, and 3 mm MgSO₂ prior to treatment with thermolysin (10 μg) and CaCl₂ (5 mm) for 15 min on ice. EDTA was added to stop the reaction and plastids pelleted. All import samples were resolved by SDS-PAGE (16% (w/v) Acrylamide SDS-PAGE gels or 16.5% (w/v) Acrylamide Tricine SDS-PAGE as described in (Schägger, 2006 (link)), dried, and exposed to a phosphor-imaging screen for 24 h.