We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes, lysosome-related organelles (e.g., melanosomes and platelet dense bodies) and specific domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Sorting is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats (e.g., clathrin coats). Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA proteins, and the heteropentameric retromer complex. Proper sorting requires the function of additional components of the trafficking machinery that mediate vesicle tethering and fusion, such as the multisubunit tethering complex GARP. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, as well as investigating human diseases that result from genetic defects of these proteins (e.g., Hermansky-Pudlak syndrome; neurodegenerative and neurodevelopmental disorders). AP-1, AP-2, and AP-3 are clathrin-associated adaptor complexes that recognize two types of sorting signal referred to as tyrosine-based and dileucine-based signals. Previous studies from our laboratory showed that tyrosine-based signals bind to the mu1, mu2 and mu3 subunits, whereas dileucine-based signals bind to a combination (i.e., a hemicomplex) of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, from the corresponding AP complexes. A major goal of our work this past year was the analysis of the role of signal-adaptor interactions in polarized sorting in neurons. Neurons are polarized into dendrites, soma and axons. The plasma membrane of each of these domains possesses a distinct set of transmembrane proteins, including receptors, channels, transporters and adhesion molecules. We hypothesized that sorting to these domains was mediated by interaction of sorting signals with AP complexes. Our studies showed that the cytosolic tails of various transmembrane receptors, including the transferrin receptor (TfR), the Coxsackie virus and adenovirus receptor (CAR), and the glutamate receptor proteins mGluR1, NR2A and NR2B, all have information leading to the sorting of these proteins to the somatodendritic domain of hippocampal neurons. In the case of TfR and CAR, this information occurs in the form of tyrosine-based sorting signals. Protein interaction analyses showed that the tails of these receptors bind to the mu1A subunit of AP-1. Dominant-negative interference and RNAi approaches demonstrated that interaction of cytosolic tails with AP-1 was responsible for somatodendritic sorting. Sorting involved exclusion of the receptor proteins from transport carriers destined for the axonal domain at the level of the soma. Interference with AP-1-dependent somatodendritic sorting caused defective maturation of dendritic spines and decreased the number of excitatory synapses. Recently, mutations in the sigma1A and sigma1B subunits of AP-1 were shown to be the cause of two syndromic mental retardation disorders known as MEDNIK syndrome and Fried syndrome, respectively. Unlike mu1A, sigma1A and sigma1B recognize dileucine-based sorting signals. Our findings suggest that the MEDNIK and Fried syndromes may arise from failure to sort dileucine-containing cargos to the somatodendritic domain of specific neuronal populations. Together with previous findings in epithelial cells, our recent results establish AP-1 as a global regulator of polarized sorting in various cell types. Structural studies provided valuable insights into the mechanisms by which AP complexes recognize sorting signals. In collaboration with James Hurley (NIDDK, NIH), we elucidated the mechanism by which the small GTPase Arf1 recruits the AP-1 complex to membranes and induces a conformational change that allows AP-1 to bind both tyrosine-based and dileucine-based sorting signals. X-ray crystallographic analysis of Arf1 in complex with the AP-1 core showed that two molecules of Arf1-GTP bind to distinct sites on the gamma and beta1 subunits of AP-1. The AP-1 core in this complex is in the open conformation that exposes binding sites for tyrosine-based and dileucine based sorting signals. The structure reveals how Arf1-induced conformational activation couples membrane recruitment to sorting-signal recognition. In addition to AP-1, we also studied the mechanisms of signal recognition by the related AP-3 complex. In collaboration with the group of Michael Marks (University of Pennsylvania School of Medicine), we showed that recognition of a dileucine-based sorting signal in the cytosolic tail of the oculocutaneous albinism type 2 (OCA2) protein by both AP-1 and AP-3 mediates OCA2 sorting to melanosomes. In a separate study, we solved the crystal structure of the mu3A subunit of AP-3 in complex with a tyrosine-based sorting signal. This structure revealed that the binding site on mu3A is similar to that on the mu2 subunit of AP-2. These studies contributed to the elucidation of the mechanisms by which the AP-3 complex sorts transmembrane proteins to melanosomes, thus shedding light on the pathogenesis of the pigmentation and bleeding disorder Hermansky-Pudlak syndrome type 2. Finally, in collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we uncovered the structural mechanism by which the tethering factor GARP interacts with its cognate vesicle fusion SNARE proteins. X-ray crystallographic analyses showed how the Ang2 subunit of GARP binds to the Habc domain of the Syntaxin 6 SNARE. These findings highlight a key event in the pathway by which transport vesicles emanating from endosomes fuse with the trans-Golgi network. The integrity of this pathway is essential for neuronal function and viability, and its perturbation leads to neurodegenerative movement disorders.