Figure 2.

A, Increasing concentrations of nigericin do not abolish OE17 processing in the presence of azide. Intact chloroplasts were preincubated with 0, 6, 15, or 30 μm nigericin (always with the same volume of ethanol) in the dark for 10 min on ice in the presence or absence of 30 mm azide (or an equivalent volume of water). Chloroplasts were diluted 3-fold into the import mixture to start the assay, and reactions were allowed to continue for 30 min in the light. The import assay was terminated and the samples analyzed as described for Figure 1. The standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted. B, Azide does not inactivate the uncoupling activity of nigericin. Isolated thylakoids were preincubated with 6 μm nigericin (or an equivalent volume of ethanol) and 30 mm azide (or an equivalent volume of water) for 10 min on ice in the dark. Thylakoids were then diluted 3-fold into import buffer containing 20 μm methyl viologen and 3 mm MgCl₂ to a concentration of 20 μg/mL. 9-Aminoacridine (5 μm) was added and the fluorescence was measured. Saturating red actinic light was turned on and off at 20 and 100 s; the shutter in front of the measuring beam was briefly shut at 75 s to determine if the sample chamber was light tight.