Molecular basis for the substrate specificity of plant guanine nucleotide exchange factors for ROP

Abstract 

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.

Abbreviations: At, Arabidopsis thaliana, Cdc42, cell division cycle 42, cDNA, complementary DNA, Dbl, diffuse B-cell lymphoma, DH, Dbl homology, G protein, guanine nucleotide binding protein, GDI, guanine nucleotide dissociation inhibitor, GEF, guanine nucleotide exchange factor, GNP, GppNHp, GST, glutathione-S-transferase, HVR, hypervariable region, IgG, immunoglobulin G, P-loop, phosphate binding loop, PCR, polymerase chain reaction, PH, Pleckstrin homology, PRONE, plant-specific ROP nucleotide exchanger, Rac/RAC, Ras-related C3 botulinum toxin substrate, Ras, rat sarcoma, Rho, Ras homologue, ROP, Rho of plants, Tiam1, T-cell lymphoma invasion and metastasis 1, 3D structure, three-dimensional structure

Keywords: Rho of plants, ROP/RAC, Rho insert, RopGEF, PRONE, Substrate specificity