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/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 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), hereditary spastic paraplegias (HSPs) and other neurodevelopmental disorders. ARL8 relieves SKIP autoinhibition to enable coupling of endolysosomes to kinesin-1. A major focus of the lab is on the mechanisms that drive movement of organelles within the cytoplasm. Long-range movement of organelles relies on coupling to microtubule motors, a process that is often mediated by adaptor proteins. In many cases, this coupling involves organelle- or adaptor-induced activation of the microtubule motors by conformational reversal of an autoinhibited state. This past year, we discovered that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small GTPase ARL8 on the endolysosomal membrane to couple endolysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP revealed that the C-terminal region comprising three PH domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of endolysosomes to kinesin-1 and, consequently, endolysosome movement toward the cell periphery. ARL8 relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde endolysosome transport. These findings demonstrate that SKIP is not just a passive connector of endolysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. 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. This past year, 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. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. 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. Structure of human ATG9A, the only transmembrane protein of the core autophagy machinery. We also continued our studies on autophagy. A major achievement was the resolution of the atomic structure of the transmembrane autophagy protein ATG9A. In collaboration with the groups of Anirban Banerjee (NICHD), Jiansen Jiang (NHLBI) and Jose Faraldo-Gomez (NHLBI), we succeeded in obtaining a 2.9-Angstrom resolution cryo-EM structure of human ATG9A. The structure revealed a novel fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a transmembrane lipid transporter. In addition, structure-guided molecular simulations predicted that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes. Regulation of LC3B levels by ubiquitination and proteasomal degradation. In previous work, we discovered that the autophagy protein LC3 is monoubiquitinated by the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6, resulting in proteasomal degradation of LC3B and attenuation of autophagic activity under conditions of stress. This past year, we found that LC3 monoubiquitination is reversed by the action of the deubiquitinating enzyme USP10. Silencing of USP10 reduces the levels of LC3 through increased ubiquitination and proteasomal degradation. In turn, reduced LC3 levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and increased accumulation of puromycin-induced protein aggregates. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination. The Golgi-associated retrograde protein (GARP) complex is critical for maintenance of the Golgi glycosylation machinery. The Golgi apparatus is a central hub for intracellular protein trafficking and glycosylation. In collaboration with the group of Vladimir Lupashin (University of Arkansas for Medical Sciences), this past year we found that the Golgi-associated retrograde protein (GARP) complex is critical for the maintenance of the Golgi glycosylation machinery. We observed that depletion of GARP subunits impairs the modification of N- and O-glycans and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-knockout (KO) cells exhibit reduced retention of glycosylation enzymes in the Golgi apparatus and their missorting to the endolysosomal system. These findings led us to propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery.