Molecular mechanisms regulating protein transport and targeting
Eukaryotic cells compartmentalize
biological functions in a series of membrane-bound
organelles. The unique composition of each compartment
is maintained despite the continuous movement of
proteins and lipids within the cell. To achieve that
proteins are specifically targeted to various
subcellular compartments. Furthermore, regulated protein
targeting also plays a key role in receptor recycling,
cell motility and cytokinesis (cell division).
Cells achieve protein
targeting through the use of transport vesicles equipped
with complex arrays of proteins that regulate vesicle
formation, transport, and fusion. Small Rab GTPases are
the key proteins involved in membrane traffic. Rabs
function as "address" tags of transport vesicles by
recruiting various effector proteins to the membranes.
Critical questions in understanding the roles of Rab
proteins are the identity and specificity of these
effectors. Since most Rab GTPases have multiple effector
proteins, the mechanisms and regulation of their binding
are essential for understanding of Rab functions in
membrane traffic.
The
identification of Rab binding proteins and understanding
their function has been main focus of our laboratory in
a last few years. The work in my laboratory led to
identification of the novel family of Rab binding
proteins, known as FIPs. Furthermore, we have shown that
different Rab-FIP complexes may serve as "targeting
patches", thus determining the fate of transport
vesicle. Three main projects are being currently
investigated in the laboratory: (1)
structure/function analysis of Rab-FIP protein
complexes; (2) the role of Rab-FIP complexes in
regulating protein and membrane targeting; (3)
identification of novel proteins interacting with
Rab-FIP "targeting patches". To address these
questions, several methods will be combined, including
structural studies using circular dichroism, x-ray
crystallography and NMR, immunoprecipitations,
immunofluorescence and time-lapse microscopy, mutant
analysis, permeabilized cell assays, proteomics,
affinity chromatography, and yeast-two hybrid screens.
(1) Structure/function analysis of Rab-FIP protein
complexes
Understanding the structure of proteins is the key step
in determining their function in the cell. Thus,
determining the properties of Rab and FIP interactions
has been one of the major focuses in the lab. The
current work concentrates on determining the structure
of Rab-FIP complex (collaboration with Dr. Bill Weis,
Stanford University) as well as kinetic properties of
Rab-FIP complex formation.
Structural information from above studies is
then used to analyse the role of Rab and FIP
interactions in vivo. Several microscopy assays
are used for that purpose. That includes fluorescent
energy transfer (FRET) as well as time-lapse microscopy
analysis (for cool movie depicting the fusion of
transport vesicle/tubule containing GFP-labeled FIP see
Figure 1).
(2) The role of Rab-FIP complexes in regulating
protein and membrane targeting
The
role of Rab-FIP protein complexes in regulating specific
membrane and protein targeting pathways remains to be
fully understood. My laboratory is also interested in
investigating the role of Rabs in several membrane
transport pathways.
- Regulation of receptor/transporter recycling
(insulin-dependent GLUT4 transport);
- Regulation of protein targeting in polarized epithelial
cells (apical versus basolateral endocytictargeting);
- Regulation of membrane transport during cell motility.
(3) Identification of novel proteins interacting with
Rab-FIP "targeting patches"
Work
from my laboratory suggest that Rab-FIP complexes may
function as "targeting patches" my recruiting additional
proteins to transport vesicles. Thus, identification of
these proteins is of a major interest for the lab. To
achieve that, we use a combination of proteomics and
yeast two-hybrid screens. These approaches so far
suggested that at least some of the Rab-FIP "targeting
patches" interact with molecular motor proteins and are
involved in regulating the motility of transport
vesicles along microtubule or actin "highways" (see
Figure 3).
Selected Publications
Tarbutton, E., and Prekeris, R. (2005) Functional properties of RCP and Rip11 in Rab11 function. Methods in Enzymology, In Press.
Fielding, A.B., Schonteich, E., Yu, X., Matheson, J.,Wilson, G., Xinzi, Y., Hickson, G.R.X., Srivastava, S., Baldwin, S.A., Prekeris, R., and G.W. Gould (2005) Rab11-FIP3 and Rab11-FIP4 interact with Arf6 and Exocyst to control membrane traffic during cytokinesis. EMBO J. In Press.
Wilson, G.M., Fielding, A.B., Simon, G., Yu, X., Andrews, P.D., Hames, R.S., Frey, A.M., Peden, A.A., Gould, G.W., and R. Prekeris. (2005) The FIP3 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis. Molecular Biology of the Cell. 16:849-860.
Junutula, J.R., Schonteich, E., Wilson, G.M., Peden, A.A., Scheller, R.H., and R. Prekeris (2004) Molecular characterization of Rab11 interactions with the members of family of Rab11-interacting proteins (FIPs). The Journal of Biological Chemistry. 279:33430-33437.
Peden, A.A., Schonteich, E., Chun, J., Jagath, J.R., Scheller, R.H., and R. Prekeris. (2004) The RCP-Rab11 complex regulates endocytic protein sorting. Molecular Biology of the Cell. 15:3530-3541.
Hickson, G.R., Matheson, J., Riggs, B., Maier, V., Fielding, A.B., Prekeris, R., Sullivan, W., Barr, F.A., G.W. Gould. (2003) Arfophillins are dual Arf/Rab11 bindong proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout. Molecular Biology of the Cell. In Press.
Meyers, J.M., and Prekeris, R. (2002) Formation of Mutually Exclusive Rab11 Complexes with Members of the FIP Family Regulate Rab11 Endocytic Targeting and Function. The Journal of Biological Chemistry. 277:49003-49010
Prekeris, R., Davies, J.M., and Scheller, R. (2001) Identification of a Novel Rab11/25 Binding Domain Present in Eferin and Rip Proteins. The Journal of Biological Chemistry. 276:38966-38970.
Martinez-Menarguez, JA., Prekeris, R., Oorschot, V., Scheller, R., Geuze, HJ., Slot, JW., and Klumperman, J. (2001) Peri-Golgi Vesicles Contain Retrograde but not Anterograde Proteins Consistent with the Cisternal Progression Model of Intra-Golgi Transport. The Journal of Cell Biology. 155:1213-1224.
Prekeris, R., Klumperman, J., and Scheller, R.H. (2000) A Rab11/Rip11 Protein Complex Regulates Apical Membrane Trafficking via Apical Recycling Endosomes. Molecular Cell. 6:1437-1448.
Prekeris, R., Yang, B., Oorschot, V., Klumperman, J., and Scheller, R.H. (1999) Differential Roles of Syntaxin 7 and Syntaxin 8 in Endosomal Trafficking. Molecular Biology of The Cell. 10:3891-3908.
Prekeris, R., Foletti, D.L., and Scheller, R.H. (1999) Dynamics of Tubulo-Vesicular Recycling Endosomes in Hippocampal Neurons. The Journal of Neuroscience. 19(23):10324-10337.
Foletti, D.L., Prekeris, R., and Scheller R.H. (1999) Generation and Maintenance of Neuronal Polarity: Mechanisms of Transport and Targeting. Neuron, 23:641-644.
Prekeris, R., Klumperman, J., Chen, Y.A., and Scheller, R.H. (1998) Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Recycling Endosomes. The Journal of Cell Biology. 143:957-971.
Prekeris, R., Hernandez, R.M., Mayhew, M.W., White, M.K., and Terrian D.M. (1998) Molecular Analysis of the Interactions between Protein Kinase C-e and Filamentous Actin. The Journal of Biological Chemistry. 273:26790-26798.
Prekeris, R., and Terrian, D.M. (1997) Brain Myosin V is a Synaptic Vesicle-Associated Motor protein: Evidence for a Ca2+-Dependent Interactions with the Synaptobrevin-Synaptophysin Complex. The Journal of Cell Biology. 137(7):1589-1601.
Prekeris, R., Mayhew, M.W., Cooper, J.B., and Terrian D.M. (1996) Identification and Localization of an Actin-Binding Motif That is Unique to the Epsilon Isoform of Protein Kinase C and Participates in the Regulation of Synaptic Function. The Journal of Cell Biology. 132(1):77-90.
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