Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling

The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector doma in amino acid threonine 37/35 by Clostridium difficile toxins A and B. Gluc osylation renders the Rho proteins inactive by inhibiting effector coupling To understand the functional consequences, effects of glucosylation on sub cellular distribution and cycling of Rho GTPases between cytosol and membra nes were analyzed. In intact cells and in cell lysates, glucosylation leads to a translocation of the majority of RhoA GTPase to the membranes whereas a minor fraction is monomeric in the cytosol without being complexed with the guanine nucleotide dissociation inhibitor (GDI-1). Rho complexed with G DI-1 is not substrate for glucosylation, and modified Rho does not bind to GDI-1. However, a membranous factor inducing release of Rho from the GDI co mplex makes cytosolic Rho available as a substrate for glucosylation. The b inding of glucosylated RhoA to the plasma membranes is saturable, competabl e with unmodified Rho-GTP gamma S guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa. Me mbrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites. T hus, in addition to effector coupling inhibition, glucosylation also inhibi ts Rho cycling between cytosol and membranes, a prerequisite for Rho activa tion.