Figure 10.

ALB3 integration was not inhibited by any thylakoid translocase antibodies; SCY1 integration was reduced significantly by anti-ALB3. Membranes recovered from a chloroplast lysate were incubated at 0.33 mg mL⁻¹ Chl with IgGs at 0.8 μg mL⁻¹ or anti-ALB3 50 aa serum and its prebleed at 100 μL of serum in import buffer containing 5 mm MgCl₂ (IBM) and 0.7% bovine serum albumin for 1 h on ice. Membranes were pelleted, washed, and resuspended in IBM. Treated membranes were assayed with 10× SE, 5 mm Mg-ATP, and in vitro translated precursor proteins Δtp-SCY1, pre-LHCP (in A), mLHCP (in B), iOE33, iOE23, and pre-ALB3 as designated to the left of the gels for 50 min in the light. Membranes were recovered, treated with thermolysin, and analyzed with SDS-PAGE and fluorography. A, LHCP served as a positive control for the SRP/ALB3 pathway, OE33 for the SEC1 pathway, and OE23 for the TAT pathway. Note that serum has a severe nonspecific inhibitory effect on the TAT pathway (lanes 7–9). B, ALB3 assays were conducted similarly, except that mLHCP was used in control experiments and aliquots of untreated assay membranes were loaded adjacent to thermolysin-treated membranes. DP, Degradation product; i, intermediate protein; m, mature protein; p, precursor protein.