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 often 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. In addition, sorting involves the function of other components of the trafficking machinery that mediate vesicle tethering and fusion, such as the multisubunit tethering complex GARP and cognate SNARE proteins. 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) or their exploitation by pathogens (e.g., HIV-1). An AP-1/clathrin pathway for the sorting of transmembrane receptors to the somatodendritic domain of hippocampal neuron. AP-1, AP-2, AP-3 and AP-4 are adaptor protein 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-1, AP-2 and AP-3 complexes. To date, the AP-4 has only been shown to recognize tyrosine-based signals via its mu4 subunit. A major goal of our recent work has been 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 vesicular 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. Co-assembly of viral envelope glycoproteins regulates their polarized sorting in neurons. We have recently extended our studies on the mechanisms of polarized sorting in neurons to the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus, a neuroinvasive pathogen that causes fatal human encephalitis. When analyzed individually, NiV-G is delivered to both the axonal and somatodendritic domains (i.e., it is non-polarized). In contrast, NiV-F is exclusively targeted to the somatodendritic domain by virtue of the interaction of a tyrosine-based sorting signal with the mu1A subunit of AP-1. Importantly, co-expression with NiV-G causes NiV-F to lose its somatodendritic polarity, becoming evenly distributed between the somatodendritic and axonal domains. This redistribution is due to the incorporation of NiV-F into axonal transport carriers in the presence of NiV-G. We also observed that NiV-F exhibits a faster rate of biosynthetic transport compared to NiV-G. These observations led us to propose that coordinated interactions of viral glycoproteins with the hosts sorting machinery and between themselves allow temporal and spatial regulation of their distribution between the somatodendritic and axonal domains of neurons, a process that may have implications for viral spread through synaptic contacts. Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine-based signal by AP-1. As mentioned above, AP-1 also binds dileucine-based signals by virtue of interactions with a site on the gamma-sigma1 hemicomplex. We have recently found that interactions of AP-1 with dileucine-based signals in the copper transporter ATP7B and the vesicle-SNARE VAMP4 also mediate sorting to the somatodendritic domain of rat hippocampal neurons. ATP7B localizes to the trans-Golgi network (TGN) under low copper conditions, but redistributes to the cell periphery under high copper conditions in hippocampal neurons. Furthermore, under low copper conditions ATP7B is restricted to the soma and dendrites and excluded from the axon. This somatodendritic polarity is lost upon mutation of the dileucine-based signal in ATP7B or overexpression of a dominant-negative sigma1 mutant incapable of binding such signals. High copper levels also cause loss of somatodendritic polarity. These observations indicate that copper levels regulate not only the distribution of ATP7B between the TGN and peripheral vesicles, but also the polarized sorting of this transporter between the somatodendritic and axonal domains. Mu1 subunit isoforms expand the repertoire of basolateral sorting signal recognition in epithelial cells. Some of the AP-1 subunits occur as various isoforms encoded by different genes. For example, there are two mu1 subunit isoforms (mu1A and mu1B) and three sigma1 subunit isoforms (sigma1A, sigma1B and sigma1C). What is the physiologic significance for the existence of these isoforms? Years ago, we found that mu1A is ubiquitously expressed, whereas mu1B is exclusively expressed in epithelial cells. Using advanced confocal microscopy techniques, including spinning disk, TIRF and super-resolution structured illumination microscopy, we found that mu1A and mu1B similarly localize to both the TGN and endosomes. However, each isoform was found to exhibit preferences for distinct set of signals. For example, yeast two-hybrid and GST pull-down experiments showed that the non-canonical tyrosine-based signals and clusters of acidic residues that mediate basolateral sorting of the low-density lipoprotein receptor preferentially bind to mu1B. We conclude that the existence of mu1 subunit isoforms expands the repertoire of AP-1 signal recognition in epithelial cells, allowing for efficient and regulated sorting of a broader set of cargos to the basolateral surface. Taken together, our studies in neurons and epithelial cells establish the AP-1 complex as a global regulator of polarized sorting. Defects in polarized sorting may underlie the pathogenesis of neuro/cutaneous disorders caused by mutation in sigma1 subunit isoforms, such as the MEDNIK syndrome (sigma1A), Fried/Pettigrew syndrome (sigma1B) and pustular psoriasis (sigma1C).
| Guardia, Carlos M; De Pace, Raffaella; Mattera, Rafael et al. (2018) Neuronal functions of adaptor complexes involved in protein sorting. Curr Opin Neurobiol 51:103-110 |
| De Pace, Raffaella; Skirzewski, Miguel; Damme, Markus et al. (2018) Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome. PLoS Genet 14:e1007363 |
| Pappas, Samuel S; Bonifacino, Juan; Danek, Adrian et al. (2017) Eighth International Chorea-Acanthocytosis Symposium: Summary of Workshop Discussion and Action Points. Tremor Other Hyperkinet Mov (N Y) 7:428 |
| Romano-Moreno, Miguel; Rojas, Adriana L; Williamson, Chad D et al. (2017) Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL. Proc Natl Acad Sci U S A 114:E11151-E11160 |
| FarÃas, Ginny G; Guardia, Carlos M; De Pace, Raffaella et al. (2017) BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the axon. Proc Natl Acad Sci U S A 114:E2955-E2964 |
| Chen, Yu; Gershlick, David C; Park, Sang Yoon et al. (2017) Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers. J Cell Biol 216:4141-4151 |
| Jia, Rui; Guardia, Carlos M; Pu, Jing et al. (2017) BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy 13:1648-1663 |
| Mattera, Rafael; Park, Sang Yoon; De Pace, Raffaella et al. (2017) AP-4 mediates export of ATG9A from the trans-Golgi network to promote autophagosome formation. Proc Natl Acad Sci U S A 114:E10697-E10706 |
| Pu, Jing; Keren-Kaplan, Tal; Bonifacino, Juan S (2017) A Ragulator-BORC interaction controls lysosome positioning in response to amino acid availability. J Cell Biol 216:4183-4197 |
| Bonifacino, Juan S; Neefjes, Jacques (2017) Moving and positioning the endolysosomal system. Curr Opin Cell Biol 47:1-8 |
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