Membrane Biology Section
Julie G Donaldson, PhD, Principal Investigator
Our group studies the function of the Arf GTP-binding proteins
in regulating membrane traffic and structure. A primary focus is
on Arf6, which coordinates membrane traffic and the actin cytoskeleton
at the plasma membrane (PM). In the active, GTP-bound state, Arf6
activates phosphatidylinositol 4-phosphate 5-kinase, an enzyme
that generates phosphatidylinositol 4,5-bisphosphate (PIP2), a
major PM phosphoinositide. PIP2 is abundant along the PM where
it facilitates actin polymerization and on endosomal membranes
associated with Arf6. Stimulated activation of Arf6, by expression
of a guanine nucleotide exchange factor (GEF) for Arf6, results
in the formation of protrusive ruffling and consequent formation
of macropinosomes, which are taken into the cells and the membrane
recycled back to the PM. Failure to inactivate Arf6, by GTP hydrolysis,
or to turnover the PIP2 leads to a disruption in this membrane
flow and a halt in PM ruffling. One area of research in the lab
is the identification of additional GEFs that activate Arf6 and
studies aimed at identifying factors that regulate GEF activity
in cells.
The Arf6-associated endosomal membranes contain PM proteins that
enter cells by endocytosis independently of adaptor protein 2 (AP2)/clathrin.
We have found that these non-clathrin derived endosomes contain
physiologically important PM proteins such as the major histocompatibility
complex class I (MHCI) proteins, integrins, "raft" partitioning
proteins like GPI-anchored proteins, and other regulators such
as Rac1 and src. Upon internalization, these non-clathrin-derived
early endosomes fuse with the traditional, Rab5-associated early
endosome and from there cargo can proceed to late endosomes and
lysosomes where they are degraded or be recycled back to the PM
via distinct, tubular, Arf6-associated recycling membranes. This
recycling pathway only carries non-clathrin cargo and requires
the activities of two Rab GTPases, Rab11 and Rab22. Further studies
aimed at elucidating how these endosomal membrane systems interconnect
will include identification of other components involved in these
pathways (such as Rabs, SNARES, and PIP-kinases) and the development
of in vitro endosomal fusion assays that recreate these activities.
We also are interested in understanding how clathrin-independent
endocytosis is regulated and identifying the molecular machinery
that is required for this process.
Arf6 belongs to the Arf Family of GTP binding proteins and is
70% identical to Arf1, the other Arf family member that is well
characterized. These two Arfs also exhibit remarkable structural
similarity and we have utilized this feature to create chimeras
between Arf1 and Arf6 in order to identify sequences critical for
localization and function. We have identified residues in Arf6
sequence adjacent to Switch I region that are required for protrusion
formation by Arf6. Recently, we have identified a determinant in
Arf1 that facilitates Golgi recruitment through binding to the
ER-Golgi SNARE protein Membrin. Using yeast 2-hybrid and biochemical
affinity approaches, projects have identified candidate, Arf6-
and Arf1-specific interacting proteins. Studies of these effector
proteins should assist in the discovery of how Arf6 and Arf1 carry
out their specific cellular functions.
Selected Publications:
Brown, F.D., Rozelle, A.L., Yin, H.L., Balla, T., and Donaldson,
J.G. Phosphatidylinositol-4,5-bisphosphate and Arf6-regulated membrane
traffic. J. Cell Biol., 154, 1007-1017, 2001.
Donaldson, J.G. Multiple roles for Arf6: Sorting, structuring,
and signaling at the plasma membrane, J. Biol. Chem. 278, 41573-41576,
2003.
Honda, A., Al-Awar, O.S., Hay, J.C., and Donaldson, J.G. Targeting
of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE, J. Cell
Biol. 168:1039-1051.
Naslavsky, N., Weigert, R., and Donaldson, J.G. Convergence between
non-clathrin and clathrin-derived endosomes involves Arf6 inactivation.
Mol. Biol. Cell 14, 417-431, 2003.
Naslavsky, N., Weigert, R. and Donaldson, J.G. Characterization
of a non-clathrin endocytic pathway: membrane cargo and lipid requirements,
Mol Biol Cell, 15:3542-3552, 2004.
Weigert, R., Yeung, A.C., Li, J. and Donaldson, J.G. Rab22a regulates
the recycling of membrane proteins internalized independently of
clathrin. Mol Biol Cell, 15:3758-3770, 2004.
Questions, comments and suggestions about this page may be
addressed to Julie Donaldson
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