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Departments of
*Pharmacological and Physiological Sciences,
Small GTPases of the Ypt/Rab family are involved in the regulation
of vesicular transport. Cycling between the GDP- and GTP-bound forms
and the accessory proteins that regulate this cycling are thought to be
crucial for Ypt/Rab function. Guanine nucleotide exchange factors
(GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating
proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs
and GAPs for Ypt/Rab proteins. In this article we report the
identification and initial characterization of two factors that
regulate nucleotide cycling by Ypt1p, which is essential for the first
two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP
release and GTP uptake at least 10-fold and is specific for Ypt1p.
Partially purified Ypt1p-GEF can rescue the inhibition caused by the
dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic
reticulum-to-Golgi transport. This mutant probably blocks
transport by inhibiting the GEF, suggesting that we have identified the
physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by
Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of
Ypt1p than for the GDP-bound form, and is specific to a subgroup of
exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by
deletion of two genes that encode known Ypt GAPs, GYP7
and GYP1, nor is it influenced by mutations in
SEC18, SEC17, or SEC22,
genes whose products are involved in vesicle fusion. The GEF and GAP
activities for Ypt1p localize to particulate cellular fractions.
However, contrary to the predictions of current models, the GEF
activity localizes to the fraction that functions as the acceptor in an
endoplasmic reticulum-to-Golgi transport assay, whereas the GAP
activity cofractionates with markers for the donor. On the basis of our
current and previous results, we propose a new model for the role of
Ypt/Rab nucleotide cycling and the factors that regulate this process.
Biochemistry and Molecular Biology, and
§Molecular Genetics and Cell Biology, The University of
Chicago, Chicago, Illinois 60637
S.J. and C.J.R. contributed equally to this
work.
Corresponding author: Department of
Pharmacological and Physiological Sciences, The University of Chicago,
947 East 58th Street, Box 271, Chicago, IL 60637. E-mail address:
ns15{at}midway.uchicago.edu.
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