Figure 1.

BFA binds to a complex between Sec7d and [Δ17]ARF1. (A) Proposed scheme for the inhibition of Sec7d by BFA as suggested by kinetic studies of Sec7d-catalyzed nucleotide exchange on ARF1 (15, 23). (B and C) Direct measurements of [³H]BFA binding. The sample (100 μl) contained initially 25 μM [³H]BFA (253 dpm/pmol) in buffer and was incubated at 27°C. At the indicated times, [YS]Arno-Sec7 (5 μM) and [Δ17]ARF1–GDP (10 μM) were added sequentially from concentrated stock solutions to the sample. Note the different order of protein additions between B and C. To determine the binding of [³H]BFA, 15-μl aliquots were withdrawn from the sample and filtered immediately onto cellulose filters, and the amount of [³H]BFA trapped on the filter was measured (see Materials and Methods). The amount of protein in each 15-μl aliquot was 75 pmol of [YS]Arno-Sec7 and/or 150 pmol of [Δ17]ARF1.