Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling.
The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector domain amino acid threonine 37/35 by Clostridium difficile toxins A and B. Glucosylation renders the Rho proteins inactive by inhibiting effector coupling. To understand the functional consequences, effects of glucosylation on subcellular distribution and cycling of Rho GTPases between cytosol and membranes 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 GDI-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 complex makes cytosolic Rho available as a substrate for glucosylation. The binding of glucosylated RhoA to the plasma membranes is saturable, competable with unmodified Rho-GTPgammaS guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa. Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites. Thus, in addition to effector coupling inhibition, glucosylation also inhibits Rho cycling between cytosol and membranes, a prerequisite for Rho activation.
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Institut für Pharmakologie und Toxikologie der Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Germany.
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D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004768:Enterotoxins / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D013912:Threonine; D020742:rhoA GTP-Binding Protein; D005947:Glucose