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 that govern the sorting of transmembrane proteins (cargo) to distinct compartments within the endomembrane system of eukaryotic cells. These compartments include the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) such as melanosomes and platelet dense bodies, and specialized plasma membrane domains in polarized cells like epithelial cells and neurons. Cargo transport between compartments is mediated by vesicular or tubular carriers that bud from donor membranes, traverse the cytoplasm, and fuse with target compartments. Our research focuses on the molecular machineries that mediate these trafficking events across various pathways, including endocytosis, endosome-to-plasma membrane recycling, retrograde transport to the TGN, lysosome and LRO biogenesis, autophagy, and polarized sorting. This fundamental work provides critical insights into the pathogenesis of congenital disorders of protein trafficking, including Hermansky-Pudlak syndrome (HPS), hereditary spastic paraplegias (HSPs), and other neurodevelopmental and early-onset neurodegenerative diseases. A SYS1–ARFRP1–ARL5–ARMH3–PI4KB regulates PI4P synthesis at the TGN – ARL5 is a small GTPase of the ARF family that is recruited to the TGN by the ARF-family GTPase ARFRP1, in complex with the transmembrane protein SYS1. At the TGN, ARL5 mediates the recruitment of its known effector, the GARP tethering complex, promoting SNARE-dependent fusion of endosome-derived retrograde transport carriers with the TGN. To explore additional functions of ARL5, we performed proximity biotinylation and protein interaction assays to identify novel effectors. These analyses revealed that the armadillo-repeat protein ARMH3 (C10orf76) specifically binds to the active, GTP-bound form of ARL5 and is recruited to the TGN in a manner dependent on SYS1, ARFRP1, and ARL5. Unlike GARP, ARMH3 is not required for retrograde cargo transport. Instead, ARMH3 plays a distinct role by activating phosphatidylinositol 4-kinase III beta (PI4KB), which generates the major pool of phosphatidylinositol 4-phosphate (PI4P) at the TGN. This PI4P pool supports the recruitment of the oncoprotein GOLPH3 and facilitates glycan processing at the TGN. Collectively, these findings identify a novel SYS1–ARFRP1–ARL5–ARMH3 axis that regulates PI4KB-mediated PI4P production at the TGN, expanding our understanding of lipid signaling and membrane trafficking at this organelle. Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration – In neurons, RNA granules are actively transported along axons to support local translation distal to the soma. Emerging evidence suggests that a subset of this RNA transport occurs via “hitchhiking” on lysosome-related vesicles. Over the past year, we further explored this mechanism by disrupting axonal entry of these vesicles through genetic knockout of the lysosome–kinesin adaptor BLOC-one-related complex (BORC). This approach enabled us to identify a population of axonal mRNAs that rely on lysosome-related vesicles for their transport. BORC knockout led to a significant depletion of axonal mRNAs, particularly those encoding components of the ribosome and mitochondrial oxidative phosphorylation machinery. The loss of these transcripts resulted in mitochondrial dysfunction and progressive axonal degeneration in both human induced pluripotent stem cell (iPSC)-derived neurons and mouse neurons. Pathway analysis of the depleted mRNA pool revealed mechanistic links between BORC deficiency and pathways implicated in common neurodegenerative diseases. These findings establish that mRNA transport via lysosome-related vesicles is essential for axonal homeostasis and demonstrate that disruption of this pathway contributes to axonal degeneration. Biallelic BORCS5 and BLOC1S1 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics – The BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits, named BORCS1 (also known as BLOC1S1) through BORCS8. BORC associates with the cytosolic surface of lysosomes, where it facilitates the recruitment of the small GTPase ARL8 and kinesin-1 and -3 microtubule motors. This recruitment promotes the anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and toward distal axons in neurons. In previous work, we demonstrated that biallelic variants in BORCS8 cause an early-infantile neurodegenerative disorder. Over the past year, through two independent collaborations—one with Niccolò Mencacci and colleagues at Northwestern University, and another with Adeline Vanderver and colleagues at the Children’s Hospital of Philadelphia—we identified additional patients with biallelic variants in two other BORC subunits: BORCS5 and BLOC1S1. These patients, all children, presented with a spectrum of neurodevelopmental abnormalities including global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facial features, optic atrophy, leuko-axonopathy with hypomyelination, and other neurodegenerative features predominantly affecting supratentorial brain regions. Cellular studies revealed that the pathogenic variants in BORCS5 and BLOC1S1 result in reduced protein expression, impaired assembly with other BORC subunits, and/or compromised ability to mediate lysosome translocation to the cell periphery. Together, these findings establish BORCS5 and BLOC1S1 as novel genetic loci for early-infantile neurodegenerative disorders and underscore the essential role of BORC and lysosomal dynamics in the development and maintenance of the central nervous system. AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia – Hereditary spastic paraplegia (HSP) comprises a group of neurogenetic disorders marked by progressive spasticity predominantly affecting the lower limbs. We collaborated with Christopher Grunseich, Kenneth Fischbeck (NINDS), Guida Landouré (University of Bamako, Mali), and colleagues to investigate a Malian family with clinical features consistent with childhood-onset complicated HSP. Neurological assessment revealed lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI demonstrated corpus callosum thinning along with cortical and spinal cord atrophy, while EEG in the index patient showed a slow background pattern. Whole-exome sequencing identified a homozygous missense variant in the gene encoding the adaptor protein complex 2 alpha-2 subunit (AP2A2). Functional studies in patient-derived neurons showed reduced AP2A2 expression, impaired transferrin receptor (TfR) endocytosis, and shortened axon initial segments. Furthermore, the AP2A2 variant disrupted binding to accessory proteins, indicating a mechanistic defect in clathrin-mediated endocytosis. These findings establish AP2A2 as a novel genetic entity associated with HSP, broadening the molecular spectrum of the disease and enhancing the precision of genetic diagnosis. A missense variant in the AP1S1 gene as a causal genetic lesion for MEDNIK syndrome – MEDNIK syndrome is a rare autosomal recessive disorder caused by pathogenic variants in the AP1S1 gene, which encodes the σ1A subunit of the adaptor protein complex 1 (AP-1), essential for intracellular protein trafficking. While previous cases linked nonsense, frameshift, and splice-site AP1S1 variants to classic MEDNIK features—including intellectual disability, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma—recent reports described two AP1S1 missense variants (c.269T>C and c.346G>A) associated with isolated enteropathy. Here, we report two patients harboring the AP1S1 c.269T>C (sigma1A L90P) variant who presented with full MEDNIK syndrome, thereby redefining its clinical spectrum. Functional analyses revealed that the sigma1A L90P variant fails to assemble into the AP-1 complex, cannot bind [DE]XXXL[LI] sorting motifs, and behaves as a loss-of-function allele. These findings confirm that all known pathogenic AP1S1 variants—including missense alleles—can cause complete MEDNIK syndrome, broadening our understanding of its molecular pathogenesis.