Quantitative GTPase affinity purification identifies Rho family protein interaction partners

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, RhoD) depending on their nucleotide loading state. We use Stable isotope labeling by Amino acids in cell culture (SILAC) and label free quantification (LFQ). Our interaction network contains many new proteins, reveals highly promiscuous binding of several effectors and mirrors evolutionary relationships of Rho GTPases.