The retinal pigment epithelium (RPE) performs key support functions for photoreceptors and the neural retina that depend on the polarized distribution of plasma membrane (PM) transporters and nutrient/adhesion receptors between apical and basolateral PM domains. Some of these transporters, e.g. Na,K-ATPase, have an opposite distribution to that found in other body epithelia (i.e. apical in RPE basolateral in other body epithelia), while other transporters have similar localizations in RPE and other epithelia (e.g. the lactate transporters MCT3 and MCT4). How does RPE acquire and maintain its specialized polarity phenotype? Our central hypothesis is that different epithelia organize their polarized trafficking machineries differently, in a tissue-specific manner. Indeed, we recently reported that RPE cells lack a key clathrin adaptor, AP1B, that sorts basolateral PM proteins in most body epithelia; this explains the reversed apical polarity or non-polarized distribution of several RPE PM Proteins (e.g. the coxsackie adenovirus receptor (CAR) and neural adhesion molecule (NCAM)). Furthermore, during the past grant cycle we discovered several basolateral sorting signals in RPE plasma membrane proteins, including two strong basolateral sorting signals in the lactate transporters MCT3 and MCT4 that explain their consistent basolateral distribution in various epithelia. These discoveries, together with our recent demonstration that clathrin is a key regulator of protein traffic to the basolateral PM set the stage for the research plan in this proposal. Specific aims 1 and 3 take advantage of cutting edge microscopic equipment and innovative live imaging protocols we recently developed to study for the first time the trafficking routes of RPE. These studies aim to elucidate the biosynthetic and recycling routes followed by apical and basolateral PM proteins in RPE and their regulation by trafficking signals, the cytoskeleton and various components of RPE's trafficking machinery. Specific aim 2 will study in detail the function of clathrin adaptors expressed by RPE cells and their interaction with our recently discovered basolateral sorting signals using yeast 2 hybrids and yeast 3 hybrid assays. We anticipate that the information provided by these studies will contribute important insights on RPE's physiology and pathology and will help devise improved therapeutic strategies for the treatment of retinal diseases, e.g. macular edema.