The Section on Intracellular Protein Trafficking investigates the molecular mechanisms that determine the sorting of integral membrane proteins in the endosomal-lysosomal system. Sorting processes such as rapid internalization from the plasma membrane, targeting to lysosomes and delivery to the basolateral plasma membrane of polarized epithelial cells are all mediated by interactions between signals in the cytosolic domains of integral membrane proteins and adaptor complexes associated with the cytosolic face of membranes. We have been particularly interested in a type of sorting signal referred to as """"""""tyrosine-based"""""""" based. These signals are recognized by the medium (m) subunits of at least three adaptor complexes named AP-1, AP-2 and AP-3. This past year, we completed a study aimed at determining if each of these adaptor complexes recognizes different subsets of tyrosine-based sorting signals. Combinatorial interaction analyses performed using the yeast two-hybrid system revealed that, indeed, the m1, m2 and m3A subunits of the AP-1, AP-2 and AP-3 complexes, respectively, exhibit distinct preferences for residues surrounding the critical tyrosine. The differential recognition of tyrosine-based signals by the adaptor complexes might underlie the involvement of such signals in various sorting processes. In other studies, we demonstrated that AP-3 binds to membranes in a reaction which is regulated by the small GTP-binding protein, ARF1. In addition, AP-3 was found to bind the coat protein clathrin, suggesting that AP-3 function may be dependent on clathrin. In collaboration with William Gahl (Heritable Disorders Branch, NICHD), we identified mutations in the b3A subunit of AP-3 in a subset of patients with Hermansky-Pudlak syndrome (HPS), a human disease characterized by reduced pigmentation of the eyes and skin, and blood platelet dysfunction. This is the first human disease shown to be caused by mutations in a component of the machinery involved in recognition of cytosolic sorting signals. The symptomatology of HPS supports the notion that AP-3 is involved in the biogenesis of lysosome-related organelles such as melanosomes and platelet dense granules. Finally, we have identified a novel adaptor medium chain named m1B which is specifically expressed in polarized epithelial cells and which might be involved in protein sorting to the basolateral plasma membrane of polarized epithelial cells.
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