(A) Localization of GFP-tagged Sec7, sec7-1, and sec7-4 after 20 min incubation at restrictive temperature (37°C).
(B) Localization of GFP-sec7-1 in cells overexpressing Ypt1 at both permissive (30°C) and restrictive (35°C) temperatures.
(C) Activation of Arf1 (measured via tryptophan fluorescence) by 100 nM WT Sec7f, sec7-4f, or sec7-4f in the presence of 500 nM activated Ypt31. Dark lines represent the average of 3 normalized reactions; lighter shaded areas represent the corresponding 95% confidence intervals (CI); dashed lines represent data not captured but inferred from curve-fitting.
(D) Quantification of Arf1 activation rates from curves in (C). Data for WT Sec7f + Ypt31 is included for comparison. The activation curve for this sample is not shown in (C) because it was acquired using a different concentration (30 nM) of the GEF. Error bars represent 95% CIs for n=3.