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. 2000 Aug 22;97(18):9913–9918. doi: 10.1073/pnas.170290597

Figure 2.

Figure 2

Effect of point mutations in Sec7d and [Δ17]ARF1–GDP on the dissociation rate of BFA from the abortive [Δ17]ARF1–GDP–Sec7d complex. (A) Time course of BFA dissociation. In a preliminary step, Sec7d (5 μM) was incubated for 10 min at 27°C with 10 μM [Δ17]ARF1–GDP/20 μM GDP/25 μM [3H]BFA. At zero time, 300 μM unlabeled BFA was added and the kinetics of [3H]BFA dissociation was followed by measuring the bound radioactivity in 15-μl aliquots. The Sec7d used were Gea2p-Sec7 (■); [YS]Arno-Sec7 (▾); [DM]Arno-Sec7 (▴); and [YSDM]Arno-Sec7 (●). (Inset) Time course of BFA dissociation from the abortive complex with [YSDM]Arno-Sec7 (●) on a different time scale as well as the dissociation of BFA from the abortive complex with [YSDM-E156K]Arno-Sec7 (○). (B) Conditional effect of the H80A mutation of [Δ17]ARF1–GDP on the dissociation rate of BFA. The time course of [3H]BFA dissociation from various abortive [Δ17]ARF1–GDP–Sec7d complexes carrying the indicated mutations in [Δ17]ARF1 and in Arno-Sec7 was measured as in A. The time constant, τoff(1/e), for BFA dissociation was determined by fitting the experimental points with a single exponential. Error bars show the standard error for two to four independent experiments. Note that the H80A mutation of [Δ17]ARF1–GDP slowed down the dissociation of BFA only when Sec7d bore the DM mutation.