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Title
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Identification of the catalytic domains and their functionally critical arginine residues of two yeast GTPase-activating proteins specific for Ypt/Rab transport GTPases.
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Authors
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S.Albert,
E.Will,
D.Gallwitz.
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Ref.
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EMBO J, 1999,
18,
5216-5225.
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PubMed id
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Abstract
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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 have
identified the catalytic domain within the C-terminal halves of two yeast
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 GTP 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 active fragments (143 and
42 microM, respectively) indicate that the affinities of those GAPs for their
substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five
invariant arginine residues; substitutions of only one of them (R343 in Gyp1p
and R458 in the analogous position of Gyp7p) rendered 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 catalytic arginine ('arginine finger') in trans to
accelerate the GTP hydrolysis rate of the transport GTPases.
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