Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases

Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation. We ha ve identified the catalytic domain within the C-terminal halves of two yeas t GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases, The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GT P hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively. The K-m values for the Gyp1p and Gyp7p activ e fragments (143 and 42 mu M, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp 1p and Gyp7p contain five invariant arginine residues; substitutions of onl y one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) r endered the GAPs almost completely inactive. We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a cataly tic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.