Regulation of GTP Hydrolysis on ADP-ribosylation Factor-1 at the
Golgi Membrane*
Edith
Szafer,
Miriam
Rotman, and
Dan
Cassel
From the Department of Biology, Technion-Israel Institute of
Technology, Haifa 32000, Israel
The interaction of the coatomer coat complex with
the Golgi membrane is initiated by the active, GTP-bound state of the
small GTPase ADP-ribosylation factor 1 (ARF1), whereas GTP hydrolysis triggers coatomer dissociation. The hydrolysis of GTP on ARF1 depends
on the action of members of a family of ARF1-directed GTPase-activating
proteins (GAPs). Previous studies in well defined systems indicated
that the activity of a mammalian Golgi membrane-localized ARF GAP
(GAP1) might be subjected to regulation by membrane lipids as well as
by the coatomer complex. Coatomer was found to strongly stimulate
GAP-dependent GTP hydrolysis on a membrane-independent mutant of ARF1, whereas we reported that GTP hydrolysis on wild type,
myristoylated ARF1 loaded with GTP in the presence of
phospholipid vesicles was coatomer-independent. To investigate the
regulation of ARF1 GAPs under more physiological conditions, we studied
GTP hydrolysis on Golgi membrane-associated ARF1. The activities at the
Golgi of recombinant GAP1 as well as coatomer-depleted fractions from
rat brain cytosol resembled those observed in the presence of
liposomes; however, unlike in liposomes, GAP activities on Golgi
membranes were approximately doubled upon addition of coatomer. By
contrast, endogenous GAP activity in Golgi membrane preparations was
unaffected by coatomer. Cytosolic GAP activity was partially reduced
following immunodepletion of GAP1, indicating that GAP1 plays a
significant although not exclusive role in the regulation of GTP
hydrolysis at the Golgi. Unlike the activities of the mammalian proteins, the Saccharomyces cerevisiae Glo3 ARF GAP
displayed activity at the Golgi that was highly dependent on coatomer.
We conclude that ARF GAPs in themselves can efficiently stimulate GTP
hydrolysis on ARF1 at the Golgi, and that coatomer may play an
auxiliary role in this reaction, which would lead to an increased cycling rate of ARF1 in COPI-coated regions of the Golgi membrane.
*
This work was supported by a grant from the Israel Science
Foundation and by the Fund for Promotion of Research (Technion, Haifa,
Israel).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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