Protein Trafficking In The Endosomal-Lysosomal System

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Our laboratory 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, cytotoxic granules), 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 vesicular or tubular carriers that bud from a donor compartment, translocate through the cytoplasm, and 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 for packaging into transport carriers, (2) microtubule motors and organelle adaptors that drive movement of transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of transport carriers to acceptor compartments. We study these machineries in the context of different intracellular transport pathways, including endocytosis, recycling from endosomes to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, autophagy, and polarized sorting in epithelial cells and neurons. Knowledge gained from this fundamental 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), hereditary spastic paraplegias (HSPs) and other neurodevelopmental disorders. The AP-4-accessory protein RUSC2 couples ATG9A-containing vesicles to the microtubule motor kinesin-1 - This past year, we continued our work on the AP-4 complex, which mediates export of the autophagy-related protein 9A (ATG9A) into transport vesicles budding from the TGN, and which is mutated in some types of HSP. We found that the AP-4-accessory protein RUSC2 couples ATG9A-containing vesicles to the plus-end-directed microtubule motor kinesin-1. These findings uncovered a mechanism for the peripheral distribution of ATG9A-containing vesicles involving the function of RUSC2 as a kinesin-1 adaptor. RUFY3 is an ARL8 effector that couples endolysosomes to the microtubule motor dynein-dynactin - The small GTPase ARL8 associates with endolysosomes, leading to the recruitment of several effectors that couple endolysosomes to kinesins for anterograde transport along microtubules, and to tethering factors for eventual fusion with other organelles. This past year, we identified RUFY3 as novel ARL8 effector that couples endolysosomes to dynein-dynactin for retrograde transport along microtubules. This function of RUFY3 in retrograde transport contributes to the juxtanuclear redistribution of endolysosomes upon cytosol alkalinization. These findings highlight the role of ARL8 in the control of not only anterograde but also retrograde endolysosome transport. SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum - In addition to coupling to microtubule motors, interactions with other organelles also regulate the movement of endolysosomes within the cytoplasm. In this regard, we found that the sorting nexin protein SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. The positioning and movement of endolysosomes within the cell is thus the result of a balance between movement driven by microtubule motors and immobilization by tethering to the ER. Decreased axonal endolysosomal motility in a human iPSC-derived inducible neuronal model of NPC1 disease - Niemann-Pick disease, type C1 (NPC1) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the NPC1 gene, and characterized by impaired cholesterol homeostasis. Mutations in NPC1 lead to deficient transport and accumulation of unesterified cholesterol in endolysosomal compartments and progressive neurodegeneration. This past year, we contributed to the characterization of a novel human iPSC-derived, inducible neuronal model of NPC1 developed in the laboratory of Forbes D. Porter (NICHD, NIH). We found that cholesterol accumulation decreases the motility of axonal endolysosomes in these neurons, and that extraction of cholesterol with 2-hydroxypropyl-beta-cyclodextrin remobilizes them. These findings shed light on the pathological mechanisms contributing to neuronal degeneration in NPC1. ATG9A enables lipid mobilization from lipid droplets - ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the autophagosome membrane. We found that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans (the latter in collaboration with Andy Golden, NIDDK, NIH). Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes. Autophagy-associated immune dysregulation in a patient with mutations in ATG9A - We also contributed to a study by Peter Williamson (NIAID, NIH) and Erwin Gelfand (National Jewish Health, Denver, CO) reporting a patient with compound heterozygous mutations in ATG9A. The patient exhibited hyperplastic proliferations of T and B cells in lung and brain, and defects in lymphocyte memory cell populations after developing an infection with Epstein-Barr virus (EBV). These defects were corrected after treatment with the mTORC inhibitor rapamycin. These results point to a novel role of ATG9A and autophagy in lymphocyte biology and provide an example of how genetic studies may suggest effective specific therapeutic interventions. Transcytosis and trans-synaptic retention by postsynaptic ErbB4 underlie axonal accumulation of NRG3 - This past year, we also collaborated with the laboratory of Andres Buonanno (NICHD, NIH) to investigate the mechanisms by which Neuregulin 3 (NRG3) localizes to the axon. We found that pro-NRG3 undergoes proteolytic cleavage by BACE1 at the TGN to generate mature NRG3. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported into axons via transcytosis. Lastly, by a mechanism we denote "trans-synaptic retention," NRG3 accumulates at presynaptic terminals by stable interaction with its receptor ErbB4 on postsynaptic GABAergic interneurons. We propose that trans-synaptic retention may account for polarized expression of other neuronal transmembrane ligands and receptors.