Epithelia consist from polarized cells that are capable of selectively transporting substances across epithelial monolayer. This selective transport is achieved by the partitioning of the plasma membrane into distinct domains: apical and basolateral, with both of these plasma membrane compartments having distinct lipid and protein compositions. Since the fidelity of trans-epithelial protein transport is crucial to a variety of epithelial functions, epithelial cells have developed complicated mechanisms to ensure correct transport of proteins. Rab11 GTPases are the members of small monomeric GTPase super-family that has been implicated in regulating endocytic membrane transport. Rab GTPase work by recruiting various effector proteins to the distinct cellular compartments. Thus, understanding the role of these effector proteins is a key step in understanding the function of epithelial cell. In the last six years several Rab11-binding proteins have been identified, which include Rab11 family interacting proteins, also known as FIPs. Works from several laboratories, including ours, have shown that Rip11/FIP5 member of FIP family regulate polarized protein transport in epithelial cells. Furthermore, it was shown that FIPs act as scaffolding factors allowing the assembly of specific sorting/transport complexes required for the epithelial protein traffic. Based on recently published results and on preliminary data, we propose the following hypotheses. First, that Rip11/FIP5 mediates protein transport to apical plasma membrane. Second, that Rip11/FIP5 regulates apical protein transport by sequential recruiting of sortin nexin 18 (SNX18) and kinesin II to the endocytic membranes. Thus, the main goal of this proposal is to further characterize Rip11/FIP5-SNX18 and Rip11/FIP5-Kinesin II complex formation and determine their role on epithelial transport. I propose three different aims designed to test these hypotheses. In the aim #1 we will analyze the role of Rip11/FIP5 in regulating apical protein transport. We will use combination of tet-inducible protein knock-down, fluorescence imaging and in vivo transport assays. In the aim #2 and aim #3 we will characterize the binding of Rip11/FIP5 to SNX18 and Kinesin II. We will use the combination of immunoprecipitations, glutathione bead pull-down, yeast two-hybrid and isothermal calorimetry assays. In addition, in aim #2 and aim #3 we will determine the roles of Rip11/FIP5-binding proteins in regulating apical and basolateral membrane traffic. To that end we will use the combination of tet- inducible protein knock-down, fluorescence imaging, site-directed mutagenesis and in vivo transport assays.
The goal of this project is to understand the molecular mechanisms of Rab11-dependent polarized membrane traffic and define the membrane traffic steps that are regulated by each Rab11-Rip11/FIP5 protein complex. Furthermore, this project will also identify the molecular machinery mediating Rab11-dependent endocytic protein sorting. These data will advance our understanding, both conceptually and mechanistically, of the machinery governing protein targeting in epithelial cells. To elucidate the mechanisms of protein targeting is of major importance because the defects in this process cause a variety of pulmonary (cystic fibrosis) and renal (nephrogenic diabetes insipidus) disorders. Thus, new insights into the mechanisms of polarized membrane traffic may lead to the identification of new drug targets as well as new therapeutic approaches aimed at the identification and treatment of membrane traffic disorders.
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