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 proteins associated with the cytosolic face of membranes. Two major families of sorting signals, referred to as tyrosine-based and dileucine-based, have been described. In previous work, we have demonstrated that tyrosine-based signals are recognized by the medium (mu) subunits of four adaptor protein (AP) complexes named AP-1, AP-2, AP-3 and AP-4. This past year, we have discovered that a subset of dileucine signals preceded by a large cluster of acidic amino acids are recognized by the GGAs, a novel family of adaptor proteins associated with the trans-Golgi network in an ARF-dependent fashion. Three GGAs (GGA1, GGA2 and GGA3) have been described in humans. The three proteins have a modular structure consisting of VHS, GAT, hinge and GAE domains. We have found that the VHS domain is directly responsible for the recognition of the acidic cluster-dileucine signals. These signals are found in the cytoplasmic domains of intracellular sorting receptors such as the mannose 6-phosphate receptors that carry lysosomal hydrolases from the trans-Golgi network to lysosomes. We found that expression of a dominant-negative GGA construct blocks exit of mannose 6-phosphate receptors from the trans-Golgi network, thus demonstrating that the GGAs mediate their sorting. We have also described another type of adaptor-related protein in humans, named stonin 1 and stonin 2. These proteins are structurally related to the medium subunits of AP complexes but do not bind either tyrosine- or dileucine-based signals. We have obtained evidence, however that they are involved in endocytosis, probably by serving as linkers between the membrane-bound protein synaptotagmin and Eps15 and intersectin, two components of the endocytic machinery. Current work is aimed at determining the roles of AP complexes, GGAs and stonins in normal cell physiology, and to investigate the possibility that defects in these proteins underlie some lysosomal diseases.
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