Fig. 8: PpRopGAP1 exhibits a higher binding capacity with PpROP4 but a much lower GAP activity than PpREN in vitro.

a PpRopGAP1 interacts with constitutively active (CA) PpROP4 but not the WT or dominant-negative (DN) forms in yeast-two-hybrid assays. 2 SD, nonselective Trp-/Leu- synthetic dropout medium. 4 SD, selective Trp-/Leu-/His-/Ade- medium. b The CRIB domain-containing N-terminal portion (Nter, 1-193) and the GAP domain-containing C-terminal portion (Cter, 194-469) of PpRopGAP1 can bind active PpROP4 separately. The binding of Cter with active PpROP4 is abolished by the activity-deficient R239L mutation. c Pull-down analyses of PpRopGAP1-PpROP4 interactions in vitro. Recombinant Glutathione S-transferase (GST)-fused PpROP4(CA) or PpROP4(DN) was purified and pulled down by maltose binding protein (MBP)-tagged WT or mutant PpRopGAP1. Coomassie blue-stained SDS-PAGE gel after pull-down is shown. d Quantification of the relative amounts of GST-PpROP4(CA) pulled down by WT and mutant MBP-PpRopGAP1. e Pull-down analyses and quantification of PpREN-PpROP4CA interactions in vitro. GST-fused PpROP4(CA) was purified and pulled down by WT or mutant forms of MBP-PpREN. Citrine and RopGAP1 were used as negative control and positive control, respectively. ROP4CA was detected with an anti-GST antibody via western blotting. f The GTPase activity of PpROP4 stimulated by PpRopGAP1 and AtRopGAP1. Relative fluorescence intensity generated by excess GTP after GTPase reactions were measured. Each reaction contained 0.25 µM PpROP4. g GTPase activity of PpROP4 stimulated by PpREN, PpREN(R204L), and PpRopGAP1. Note that PpREN is highly active and can stimulate GTP hydrolysis at a concentration around ten-fold lower than PpRopGAP1. Each experiment in (c–g) was performed with three replicates. Data in (d–g) are presented as mean values ± SD with individual points shown in (d, e). Source data are provided as a Source Data file.