Skip to main content
. 2019 Oct 24;8:e50471. doi: 10.7554/eLife.50471

Figure 7. RhoGDI extracts both inactive and active RhoGTPases from membranes in vitro.

(A) Wash off experiments: prenylated Cdc42 in both inactive (Cdc42:GDP) and constitutively-active (Cdc42G12V:GTPγS) states were reconstituted on SLBs and washed in presence of 5 μM GDI. Time lapse images at selected time points are shown; (B) Quantification of wash off experiments in which the concentration of GDI was titrated between 0 and 20 μM; (C) Koff values obtained for inactive and constitutively-active Cdc42G12V fitting the decay curves with a monoexponential decay function are plotted against GDI concentration. Extraction rates were fitted with a hyperbolic function; fitting parameters Kd and Vmax are reported in table G; (D) Ratio of Koff obtained for inactive and constitutively-active Cdc42G12V at the same GDI concentration; (E–F, H) Same as in C-D and G for inactive (Rho:GDP) and constitutively-active (RhoG14V:GTPγS) Rho. Scale bar 10 μm.

Figure 7—source data 1. RhoGDI extracts both inactive and active RhoGTPases from membranesin vitro.
elife-50471-fig7-data1.xlsx (138.6KB, xlsx)

Figure 7.

Figure 7—figure supplement 1. Nucleotide state of constitutively-active RhoGTPase variants after purification.

Figure 7—figure supplement 1.

The nucleotide state of Cdc42G12V (A), RhoG14V (B), Cdc42Q61L (D) and RhoQ63L (E) was assessed by reversed phase chromatography monitoring absorbance at 254 nm. A sample containing nucleotide standards was run on the same day of the samples (C for A and B; F for D and E).
Figure 7—figure supplement 2. RhoGDI extracts both inactive and active RhoGTPases from membranes in vitro.

Figure 7—figure supplement 2.

(A) Wash off experiments: prenylated Cdc42 in both inactive (Cdc42:GDP) and constitutively-active (Cdc42Q61L:GTP) states were reconstituted on SLBs and washed in presence of 5 μM GDI. Time lapse images at selected time points are shown; (B) Quantification of wash off experiments in which the concentration of GDI was titrated between 0 and 20 μM; (C) Koff values obtained for inactive and constitutively-active Cdc42Q61L fitting the decay curves with a monoexponential decay function are plotted against GDI concentration. Extraction rates were fitted with a hyperbolic function; (D) Ratio of Koff obtained for inactive and constitutively-active Cdc42Q61L at the same GDI concentration; (F) Same as in C-D for inactive (Rho:GDP) and constitutively-active (RhoQ63L:GTP) Rho. Scale bar 10 μm.
Figure 7—figure supplement 2—source data 1. RhoGDI extracts both inactive and active RhoGTPases from membranesin vitro.
Figure 7—figure supplement 3. Comparison of G12V and Q61L constitutively-active RhoGTPases.

Figure 7—figure supplement 3.

Comparison of Koff values obtained for the two constitutively-active variants of Cdc42 (Cdc42G12V and Cdc42Q61L, (A and C) and Rho (RhoG14V and RhoQ63L, (B and D) from wash off experiments in presence of either WT (A–B) or E158/9Q GDI (C–D). Extraction rates were fitted with a hyperbolic function.
Figure 7—figure supplement 4. Comparison of bovine and Xenopus RhoGDI in their ability to extract both inactive and active RhoGTPases from synthetic membranes.

Figure 7—figure supplement 4.

(A) Koff values obtained for inactive (Cdc42:GDP) and constitutively-active (Cdc42Q61L:GTP) Cdc42 at different bovine GDI concentrations. Extraction rates were fitted with a hyperbolic function; (A’) Ratio of Koff obtained for inactive and constitutively-active Cdc42 at the same bovine GDI concentration; (B–B’) same as in A-A’ for inactive (Rho:GDP) and constitutively-active (RhoQ63L:GTP) Rho.
Figure 7—figure supplement 4—source data 1. Comparison of bovine andXenopusRhoGDI in their ability to extract both inactive and active RhoGTPases from synthetic membranes.