Our lab investigates the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes), and different domains of the plasma membrane in polarized cells such as epithelial cells and neurons. Transport of cargo between these compartments is mediated by carrier vesicles or tubules that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors and organelle adaptors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this basic research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) and hereditary spastic paraplegias (HSPs), and the exploitation of intracellular transport by pathogens such as HIV-1. AP-4 mediates export of ATG9A from the TGN to promote autophagosome formation - This past year we discovered a role for the heterotetrameric AP-4 complex in the signal-mediated export of the autophagy protein ATG9A from the TGN, contributing to the elucidation of the pathogenesis of a group of hereditary spastic paraplegias (HSPs) caused by mutations in AP-4 subunit genes. The HSPs are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in any of the four subunits of AP-4 cause an autosomal recessive form of complicated HSP referred to as AP-4 deficiency syndrome. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. To elucidate the pathogenetic mechanism, we characterized a knockout (KO) mouse for the AP4E1 gene encoding the epsilon subunit of AP-4. We found that AP-4 epsilon KO mice exhibit a range of neurological abnormalities, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, the KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Biochemical and cellular analyses identified ATG9A, the only multispanning membrane component of the core autophagy machinery, as a specific AP-4 cargo. Moreover, we found that AP-4 promotes signal-mediated export of ATG9A from the TGN to the peripheral cytoplasm, contributing to lipidation of the autophagy protein LC3B and maturation of pre-autophagosomal structures. Immunohistochemical analyses of various AP-4-deficient cell types, including non-neuronal cell lines, patient skin fibroblasts and mouse neurons, showed retention of ATG9A at the TGN and its depletion from the peripheral cytoplasm. ATG9A mislocalization was associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 epsilon KO neurons. These findings indicated that the AP-4 epsilon KO mouse is a suitable animal model for human AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder. Segregation in the Golgi complex precedes export of endolysosomal proteins in distinct transport carriers, independently of AP complexes - The studies described above demonstrated that ATG9A requires AP-4 for its signal-dependent export from the TGN. Other biosynthetic cargos destined for the endolysosomal system were also proposed to exit the TGN by interaction of sorting signals with adaptor proteins. However, there was no direct evidence for such a mechanism. Using advanced imaging methodologies, we found that this is indeed the case for the cation-dependent mannose 6-phoshate receptor (CD-MPR) and sortilin, which require interaction with the GGA adaptor proteins for export from the TGN into transport carriers bound for the endosomal system. In contrast, the transferrin receptor (TfR) and the lysosomal protein LAMP1 were found to exit the TGN independently of sorting signals and adaptor proteins. Moreover, they were transported into a different type of carrier directed towards the plasma membrane rather than endosomes and lysosomes. These proteins subsequently undergo AP-2-dependent endocytic delivery to their corresponding compartments. Strikingly, we observed that these different TGN export mechanisms are preceded by early segregation of the corresponding proteins within distinct domains of the Golgi stack by virtue of the luminal and transmembrane domains of the proteins. These findings revealed a diversity of sorting mechanisms in the Golgi complex, including early segregation in the Golgi stack prior to export into distinct populations of transport carriers. Function in the BORC complex in the regulation of lysosome movement - Another important accomplishment of the lab was the discovery the BORC complex and its role in lysosome positioning. The multiple functions of lysosomes are critically dependent on their ability to move bidirectionally along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes are mediated by kinesin and dynein motors, respectively. We recently discovered a multi-subunit complex named BORC that recruits the small GTPase ARL8 to lysosomes to promote their kinesin-dependent movement towards the cell periphery. We showed that BORC and ARL8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1B and KIF1A). Remarkably, KIF5B and KIF1B/KIF1A move lysosomes along different microtubule tracks. These findings established BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. Interaction of BORC with Ragulator controls lysosome positioning in response to amino acid availability - This past year we discovered an additional role for BORC in the response of lysosomes to amino-acid deprivation. Depletion of amino acids from the medium turns off a signaling pathway involving the Ragulator complex and the Rag GTPases, causing inactivation of the mammalian target of rapamycin complex 1 (mTORC1) kinase from the lysosomal membrane. Decreased phosphorylation of mTORC1 substrates inhibits protein synthesis while activating autophagy. Amino acid depletion also causes clustering of lysosomes in the juxtanuclear area of the cell, but the mechanisms responsible for this phenomenon were poorly understood. We found that Ragulator directly interacts with BORC, inhibiting its ability to drive lysosomes towards the cell periphery. Amino-acid depletion strengthens this interaction, explaining the redistribution of lysosomes to the juxtanuclear area. These findings demonstrated that amino acid availability controls lysosome positioning through Ragulator-dependent modulation of BORC.
| 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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