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REGULATION OF MEMBRANE
DYNAMICS AND PROTEIN TRANSPORT BY THE SEC7-DOMAIN GUANINE
NUCLEOTIDE EXCHANGE FACTORS
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Catherine L. Jackson, PhD, Head, Unit on GTPase Regulation of Membrane Traffic Ting-Kuang
Niu, PhD, Postdoctoral Fellow Elena
Smirnova, PhD, Postdoctoral Fellow Deng
Yi, PhD, Postdoctoral Fellow Peter Thomas, BS, Postbaccalaureate Fellow |
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Identification
of interacting partners of the Golgi-localized Arf GEFs in yeast and
mammalian cells Smirnova, Park, Yi The Arf GEFs of the Golgi-localized Gea/GBF
and Sec7/BIG subfamilies are large multidomain proteins. A major goal of our
laboratory is to understand the functions of the different domains of the
Gea/GBF and Sec7/BIG Arf GEFs. Given that the GEFs are soluble proteins that
must be targeted to membranes, identification of their membrane-targeting
signals and partners is an important step in understanding their function.
Within each subfamily, regions upstream and downstream of the Sec7 catalytic
domain are conserved from yeast to humans. However, the functions of these
homology regions are not known. We are carrying out two-hybrid screens with
the N-terminal and C-terminal regions of the Arf GEFs of the Gea/GBF and
Sec7/BIG subfamilies. Two-hybrid screen using BIG2, the Sec7 domain of the
mammalian homolog of Sec7p, revealed a number of partners, two of which are
human PI4P 5 kinase beta and a
putative lipase. PI4P 5 kinase beta is an interesting potential partner that
has been identified as an effector of Arf. We carried out a reverse
two-hybrid screen to determine the region of the BIG2 Sec7 domain that
interacts with PI4P 5 kinase. In finding that residues in the C-terminal
region of the Sec7 domain abolish interaction with PI4P 5 kinase, we have
narrowed down the interaction domain of PI4P 5 kinase to a 50-amino acid
region in the C-terminal portion of the catalytic domain, just upstream of
the activation loop. The activation loop contains the substrate-binding site
and is both necessary and sufficient to determine intracellular localization
of the kinase. We are also mapping the region of Arf interaction in PI4P 5
kinase, which appears to be located in a different domain of the protein than
the BIG2 Sec7 domain–interacting region. It is striking that we have
identified three lipid-modifying enzymes as binding partners of different
Sec7 domains. It appears that all three proteins bind to the C-terminal
region of the Sec7 domain. We know that this region is adjacent to a PH
domain in the We also identified subunits of two large
complexes involved in trafficking in the early secretory pathway in
two-hybrid screens with Gea2p. Using the N-terminal portion of Gea2p, we
identified Cog4p, a member of the eight-subunit COG complex that functions in
the Golgi of both yeast and mammalian cells. In the screen with the
C-terminus of Gea2p, we identified a component of TRAPP, another
Golgi-localized complex conserved in all eukaryotes from yeast to humans.
These complexes have been shown to play a role in early steps of membrane
fusion reactions before the actual fusion event, and we are now working
toward understanding the role of the Arf GEFs in this process. We have
confirmed a number of interactions by co-immunoprecipitation in yeast, in
particular Gea2p and the coat complex COPI. We are exploring other GEF-coat
interactions by two-hybrid screening, GST pulldown, and
co-immunoprecipitation in yeast and mammalian cells. Using different regions of the Gea2p protein, we
identified six trans-membrane–domain proteins as potential membrane receptors
for the Gea1p and Gea2p proteins in two-hybrid screens. Two of these partners
are Drs2p, a Golgi-localized amino-phospholipid translocase, and Gmh1p, a
Golgi-localized five-span transmembrane protein of unknown function. As is
the case for drs2, cells deleted
for GMH1 also show only a mild
effect on Gea2p. The double mutant drs2
gmh1 is viable, and, despite an effect on Gea2p localization that is more
severe than in either single mutant, some Gea2p is still able to bind to
membranes. We are testing the possibility that the other transmembrane-domain
proteins identified in two-hybrid screens with Gea2p also contribute to Gea2p
localization. Hence, the mechanism by which the large Arf GEFs such as Gea2p
are localized to membranes appears to be complex, with multiple membrane
localization determinants each contributing to the steady-state localization
of the protein in cells. Dynamics
of the GBF1 Arf GEF in mammalian cells Niu; in collaboration with
Lippincott-Schwartz, Pfeifer In collaboration with Andrea Pfeifer and
Jennifer Lippincott-Schwartz, we are using live cell imaging to study GBF1,
the mammalian homolog of Gea2p. A YFP-GBF1 fusion protein localizes to the
Golgi in mammalian cells, as previously reported. Overexpression of GBF1
confers resistance to brefeldin A (BFA), a drug that profoundly affects the
structure and functioning of intracellular organelles. Within 10 minutes of
treatment, the Golgi apparatus is completely disassembled in a normal cell,
whereas in cells overexpressing GBF1, the Golgi remains intact. YFP-GBF1 also
protects cells against the effects of BFA, indicating that this fusion
protein is functional. Remarkably, when cells are treated with BFA, GBF1 is
recruited dramatically to Golgi membranes. We have tested different mutants
in the catalytic domain of GBF1 for their response to BFA. One mutation,
E694D, is in the catalytic glutamic acid residue and reduces the rate of
exchange 400-fold in vitro. We
observed no difference in the dynamics of GBF1-E694D in cells either before
or after BFA treatment. In contrast, mutation of a residue known to affect
BFA sensitivity/resistance in other Arf GEFs without affecting catalytic
activity results in a version of GBF1 that is completely resistant to the
effects of BFA. The result indicates that GBF1 is a direct target of BFA at
the Golgi in mammalian cells. The very rapid kinetics of
association-dissociation from Golgi membranes suggests that GBF1 must
interact with localization machinery at each stage of the secretory pathway
at which it acts rather than being recruited early in the pathway and staying
associated with membranes through successive steps of the pathway. That
observation is consistent with the results described above, namely, that
numerous transmembrane partners have been identified for the GBF/GEA
subfamily of Arf GEFs. Niu TK, Pfeifer AC, Lippincott-Schwartz J,
Jackson CL. Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at
the Golgi. Mol Biol Cell 2004;Dec 22 [Epub
ahead of print]. Role
of the yeast Arf GEFs in the morphology of the yeast secretory pathway The Arf GEFs are important regulators of organelle
structure and protein trafficking both in yeast and mammalian cells. Yeast
organelles appear to differ markedly in structure and organization from those
of mammalian cells, yet most of the proteins involved in organelle structure
and trafficking, including the Arf GEFs, are highly conserved. As a first
step in identifying the structural features common to yeast and mammalian
organelles, our current work aims at determining Golgi structure in yeast by
using both live imaging and electron microscopy. Investigating both the yeast
and mammalian systems will allow us to determine which aspects of Arf GEF
function are fundamental to all eukaryotic organisms and which are unique to
their specific system. We have isolated 75 temperature-sensitive (ts)
SEC7 mutants and over 100 GEA2 ts mutants. The mutants were
generated in GFP-tagged versions of Sec7p and Gea2p and screened by
fluorescence microscopy for localization of the mutant protein at the
nonpermissive temperature. In both cases, we found a category of mutants that
showed a largely cytosolic pattern and are examining them to determine
whether they have lesions in a membrane localization domain. Other mutants
show abnormal structures at the fluorescence level. We sequenced all the
mutants (those with and without abnormal structures at the fluorescence
level) to determine the amino acids altered in the mutant proteins and are
currently examining structural changes observed in eight of the mutants at
the ultrastructural level, studies that are providing a wealth of detailed
information that cannot be obtained by light microscopy. A subset of the
mutants have only one or two amino acid substitutions in domains highly
conserved from yeast to humans. We are exploring the functions of these
domains through suppressor screens aimed at identifying potential interacting
partners of these regions of the Arf GEFs. The studies will provide
information on the molecular mechanisms that generate and maintain organelle
structure in eukaryotic cells. Cox R, Mason-Gamer RJ, Jackson CL, Segev N.
Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers. Mol Biol Cell 2004;15:1487-1505. Jackson CL. N-terminal acetylation targets
GTPases to membranes. Nat Cell Biol
2004;6:379-380. COLLABORATORS William J. Brown, PhD, Susan Ferro-Novick, PhD, Todd Graham, PhD, Jennifer Lippincott-Schwartz,
PhD, Cell Biology and Metabolism
Branch, NICHD, Andrea Pfeifer, PhD, Cell Biology and Metabolism Branch, NICHD,
Alain Rambourg, PhD, Department of Biology, CEA/Saclay, Nava Segev, PhD, For further information, contact cathyj@helix.nih.gov |
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