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Rytis Prekeris, Associate Professor

Ph.D. (1997), East Carolina University - Greenville
 

 

 

Contact Info:

Molecular Biology
University of Colorado

Rytis Prekeris, Ph.D.  Research One South
(RC1-South), Room 12112 Rytis.Prekeris@ucdenver.edu Phone: 303-724-3411

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).  

     
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(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.

  1. Regulation of receptor/transporter recycling (insulin-dependent GLUT4 transport);
  2. Regulation of protein targeting in polarized epithelial cells (apical versus basolateral endocytictargeting);
  3. 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).

 

 

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.