Figure 4—figure supplement 1. | The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

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The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

Figure 4—figure supplement 1.

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Cornell University, United States
Figure 4—figure supplement 1.
Download figureOpen in new tabFigure 4—figure supplement 1. Measurement of Sec7 GEF kinetics by liposome flotation.

2 μM myristoylated Arf1-GDP was incubated with liposomes, excess GMPPNP, and. 67 μM Sec7ΔC or GEF domain for the indicated amount of time and put on ice to stop the reaction. Lipid-bound protein (i.e. activated Arf1) was assayed by liposome flotation as described previously (Richardson et al., 2012). (A) Input fractions and float/lipid-bound fractions were analyzed by SDS-PAGE and colloidal Coomassie; float fraction load amounts were normalized to the recovered lipid fraction on the basis of fluorescence of DiR dye included in the liposomes. Note that band staining intensity correlates with molecular weight, and the GEF domain construct is ~¼ the mass of the Sec7ΔC construct. (B) Quantified band densities (circles and dashed lines) are overlaid on the reaction kinetics measured by fluorescence in Figure 4A (solid lines).

DOI: http://dx.doi.org/10.7554/eLife.12411.014