Mechanisms of polarized protein sorting in AP-1B-deficient epithelia

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Project Summary Epithelial cells carry out secretory and absorptive functions that require the polarized distribution of transporters, receptors and adhesion molecules at the apical and basolateral plasma membranes. Acquisition and maintenance of apical-basolateral polarity involves sorting of plasma membrane proteins along the biosynthetic and recycling pathways. The question of how apical and basolateral proteins in transit to the plasma membrane are sorted in endosomal compartments has been at the center of research effort for decades. An important mechanism of protein sorting is mediated by the clathrin adaptor protein AP-1B, which is a feature of epithelial cells. AP-1B recognizes cargo at discrete sorting motifs and it was initially identified as mediator of basolateral sorting. However, recent research by the applicant and others has determined that AP-1B also mediates apical sorting. Moreover, some epithelia like the kidney proximal tubule and the retinal pigment epithelium constitutively lack AP-1B. Therefore, additional mediators of polarized sorting must exist in these epithelia. This fact, and our fluid understanding of AP-1B-mediated trafficking warrants revisions of the traditional models of polarized protein sorting in epithelial cells. Filling these gaps in knowledge will reveal mechanisms that can be exploited to understand and treat pathologies where epithelial physiology is compromised. The applicant’s research program aims to characterize mechanisms of polarity in AP-1B-deficient epithelia and their role in tissue organization, homeostasis and disease. The focus of the current R35 application will be on epithelial cells of the kidney proximal tubule and retinal pigment epithelium, which lack AP-1B. We will study the roles of an endosomal sorting protein previously characterized in non-polarized cells, sorting nexin-27 (SNX27) and vesicle-associated membrane proteins (VAMPs) in polarized trafficking. We will also employ proteomics and high throughput approaches to identify additional molecular mediators of polarized trafficking. We will test whether the identified mechanisms mediate apical delivery of Megalin, a physiologically relevant surface receptor expressed in kidney proximal tubule and retinal pigment epithelium. The R35 funding mechanism is an excellent fit for achieving these goals, since it is intended to support the pursue of ambitious and flexible ideas with the intent of developing the applicant’s research and training program, rather than an individual project. At the same time, completing this proposal will lay out foundations for future advances in diagnosis, treatment, and prevention of diseases, therefore directly contributing to the mission of the National Institute of General Medical Sciences.