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The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its central loop (residues 149-179) and its core. The determinants for Nef binding include residues that directly contact AP-2 and others that stabilize the binding-competent conformation of the central loop. Residues involved in both direct and indirect interactions are required for the binding of Nef to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to interact with its binding site on Nef.

Development of the secondary palate in mammals is a complex process that can be easily perturbed, leading to the common and distressing birth defect cleft palate. Animal models are particularly useful tools for dissecting underlying genetic components of cleft palate. We describe a new cleft palate model resulting from a transgene insertion mutation. Transgene insertional mutagenesis disrupts the genomic organization and expression of the Apß1 gene located on chromosome 11. This gene encodes the ß2-adaptin subunit of the heterotetrameric adaptor protein 2 complex involved in clathrin-dependent endocytosis. Homozygous cleft palate mutant mice express no Apß1 messenger RNA or ß2-adaptin protein and die during the perinatal period. Heterozygous mice are phenotypically normal despite expressing diminished ß2-adaptin messenger RNA and protein compared with wildtype. Remarkably, the paralogous ß1-adaptin subunit of the adaptor protein 1 complex partially substitutes for the missing ß2-adaptin in embryonic fibroblasts from homozygous mutant mice, resulting in assembly of reduced levels of an adaptor protein 2 complex bearing ß1-adaptin. This variant adaptor protein 2 complex is, therefore, apparently capable of maintaining viability of the homozygous mutant embryos until birth but insufficient to support palatogenesis. Nonsyndromic cleft palate in an animal model is associated with disruption of the Apß1 gene. [ABSTRACT FROM AUTHOR]

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269 T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269 T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269 T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. KEY MESSAGES: A missense AP1S1 c.269 T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein., (© 2024. The Author(s).)

Decreased presence or activity of human SLC26A4 at the plasma membrane is a common cause of hearing loss. SLC26A4 (Pendrin) is necessary for normal reabsorption of endolymph, the fluid bathing the inner ear. We identified the μ2 subunit of adaptor protein 2 (AP-2) complex required for clathrin-mediated endocytosis as a protein-partner of SLC26A4 involved in regulating its plasma membrane abundance. We showed that, in the endolymphatic sac, where fluid reabsorption occurs, SLC26A4 is localized along the apical microvilli of mitochondria-rich cells, in contact with the endolymph, and associated with clathrin-coated pits where μ2 and AP-2 are present. Based on SLC26A4 structure, the elements involved in SLC26A4-μ2 interaction were identified and validated experimentally, allowing modeling of this interaction at the atomic level. Pharmacological inhibition of clathrin-mediated endocytosis led to an increased plasma membrane abundance of hemagglutinin-tagged SLC26A4 virally or endogenously expressed in mitochondria-rich cells. These results indicate that the SLC26A4-μ2 interaction regulates SLC26A4 abundance at the apical surface of mitochondria-rich cells.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Programmed ribosomal frameshifting is a translational recoding phenomenon in which a proportion of ribosomes are stimulated to slip backwards or forwards on an mRNA 1 , rephasing the ribosome relative to the mRNA. While frameshifting is often employed by viruses 2 , very few phylogenetically conserved examples are known in vertebrate genes and the evidence for some of these is controversial 3,4 . Here we report a +1 frameshifting signal in the coding sequence of the human gene PLEKHM2 , encoding the ARL8-dependent, lysosome-kinesin-1 adaptor protein PLEKHM2 5 . This +1 frameshifting signal, UCC_UUU_CGG, is highly conserved in vertebrates and exhibits an influenza virus-like frameshift motif with similar efficiency 6,7 . Purification and mass spectrometry of GFP-tagged trans-frame protein from cells confirms frameshifting. Structure prediction shows that the new C-terminal domain generated by this frameshift forms an alpha-helix. This additional domain relieves PLEKHM2 from autoinhibition, allowing it to move to the tips of cells via association with kinesin-1 without requiring activation by ARL8. Thus, the frameshift proteoform generates a constitutively active adaptor of kinesin-1.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition., Competing Interests: Declaration of competing interest The authors report no conflicts of interest., (Published by Elsevier Inc.)

In neurons, RNA granules are transported along the axon for local translation away from the soma. Recent studies indicate that some of this transport involves hitchhiking of RNA granules on lysosome-related vesicles. In the present study, we leveraged the ability to prevent transport of these vesicles into the axon by knockout of the lysosome-kinesin adaptor BLOC-one-related complex (BORC) to identify a subset of axonal mRNAs that depend on lysosome-related vesicles for transport. We found that BORC knockout causes depletion of a large group of axonal mRNAs mainly encoding ribosomal and mitochondrial/oxidative phosphorylation proteins. This depletion results in mitochondrial defects and eventually leads to axonal degeneration in human induced pluripotent stem cell (iPSC)-derived and mouse neurons. Pathway analyses of the depleted mRNAs revealed a mechanistic connection of BORC deficiency with common neurodegenerative disorders. These results demonstrate that mRNA transport on lysosome-related vesicles is critical for the maintenance of axonal homeostasis and that its failure causes axonal degeneration., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system., (Published by Oxford University Press on behalf of the Guarantors of Brain 2023.)

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1 ) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro , LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo , endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2 , the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis., Competing Interests: Competing Interests: The authors declare that they have no conflicts of interest.

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits., Competing Interests: Declaration of Competing Interest The authors state that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting., (© 2023. The Author(s).)

Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Spastic paraplegia 50 (SPG50) is an ultrarare childhood-onset neurological disorder caused by biallelic loss-of-function variants in the AP4M1 gene. SPG50 is characterized by progressive spastic paraplegia, global developmental delay, and subsequent intellectual disability, secondary microcephaly, and epilepsy. We preformed preclinical studies evaluating an adeno-associated virus (AAV)/AP4M1 gene therapy for SPG50 and describe in vitro studies that demonstrate transduction of patient-derived fibroblasts with AAV2/AP4M1, resulting in phenotypic rescue. To evaluate efficacy in vivo, Ap4m1-KO mice were intrathecally (i.t.) injected with 5 × 1011, 2.5 × 1011, or 1.25 × 1011 vector genome (vg) doses of AAV9/AP4M1 at P7-P10 or P90. Age- and dose-dependent effects were observed, with early intervention and higher doses achieving the best therapeutic benefits. In parallel, three toxicology studies in WT mice, rats, and nonhuman primates (NHPs) demonstrated that AAV9/AP4M1 had an acceptable safety profile up to a target human dose of 1 × 1015 vg. Of note, similar degrees of minimal-to-mild dorsal root ganglia (DRG) toxicity were observed in both rats and NHPs, supporting the use of rats to monitor DRG toxicity in future i.t. AAV studies. These preclinical results identify an acceptably safe and efficacious dose of i.t.-administered AAV9/AP4M1, supporting an investigational gene transfer clinical trial to treat SPG50.

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders., (Published by Oxford University Press 2022.)

Cutting up food into small pieces is well known to improve digestion. New work now shows that this concept also applies in the cellular world, by demonstrating that phagolysosome vesiculation promotes cell corpse degradation in Caenorhabditis elegans blastomeres., Competing Interests: Declaration of interests The authors declare no competing Interests., (Published by Elsevier Inc.)

Abbreviations: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

The transfer of endocytosed cargoes to lysosomes (LYSs) requires HOPS, a multiprotein complex that tethers late endosomes (LEs) to LYSs before fusion. Many proteins interact with HOPS on LEs/LYSs. However, it is not clear whether these HOPS interactors localize to LEs or LYSs or how they participate in tethering. Here, we biochemically characterized endosomes purified from untreated or experimentally manipulated cells to put HOPS and interacting proteins in order and to establish their functional interdependence. Our results assign Rab2a and Rab7 to LEs and Arl8 and BORC to LYSs and show that HOPS drives LE-LYS fusion by bridging late endosomal Rab2a with lysosomal BORC-anchored Arl8. We further show that Rab7 is absent from sites of HOPS-dependent tethering but promotes fusion by moving LEs toward LYSs via dynein. Thus, our study identifies the topology of the machinery for LE-LYS tethering and elucidates the role of different small GTPases in the process., Competing Interests: Declaration of interests The authors declare that there is no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inflammasome complexes are pivotal in the innate immune response. The NLR family pyrin domain containing protein 3 (NLRP3) inflammasome is activated in response to a broad variety of cellular stressors. However, a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill defined. Here, we demonstrate that NLRP3 inflammasome activators primarily converge on disruption of endoplasmic reticulum-endosome membrane contact sites (EECS). This defect causes endosomal accumulation of phosphatidylinositol 4-phosphate (PI4P) and a consequent impairment of endosome-to-trans-Golgi network trafficking (ETT), necessary steps for endosomal recruitment of NLRP3 and subsequent inflammasome activation. Lowering endosomal PI4P levels prevents endosomal association of NLRP3 and inhibits inflammasome activation. Disruption of EECS or ETT is sufficient to enhance endosomal PI4P levels, to recruit NLRP3 to endosomes and to potentiate NLRP3 inflammasome activation. Mice with defects in ETT in the myeloid compartment are more susceptible to lipopolysaccharide-induced sepsis. Our study thus identifies a distinct cellular mechanism leading to endosomal NLRP3 recruitment and inflammasome activation., (© 2022. The Author(s).)

In 1956, referring to the emerging application of electron microscopy to the study of eukaryotic cells, Keith R. Porter wrote, "For those of us who are fortunate to be part of this new development, these are days of great interest and opportunity." Those early days left us a rich legacy of knowledge on the internal organization of eukaryotic cells that provides a framework for current research on cell structure and function. In this vein, my long-time quest has been to understand how proteins and organelles travel through the cytoplasm to reach their respective destinations within the cell. This research has led us to elucidate various mechanisms of protein sorting and organelle transport and how defects in these mechanisms cause human disease.

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules., (© 2022 Jani et al.)

Adaptor protein 4 (AP-4) is a heterotetrameric complex composed of ε, β4, μ4, and σ4 subunits that mediates export of a subset of transmembrane cargos, including autophagy protein 9A (ATG9A), from the trans -Golgi network (TGN). AP-4 has received particular attention in recent years because mutations in any of its subunits cause a complicated form of hereditary spastic paraplegia referred to as "AP-4-deficiency syndrome." The identification of proteins that interact with AP-4 has shed light on the mechanisms of AP-4-dependent cargo sorting and distribution within the cell. However, the mechanisms by which the AP-4 complex itself is assembled have remained unknown. Here, we report that the alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34) binds to and stabilizes the AP-4 ε and σ4 subunits, thus promoting complex assembly. The physiological importance of these interactions is underscored by the observation that AAGAB-knockout cells exhibit reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN like those in cells with mutations in AP-4-subunit genes. These findings demonstrate that AP-4 assembly is not spontaneous but AAGAB-assisted, further contributing to the understanding of an adaptor protein complex that is critically involved in development of the central nervous system.

Neuregulins (NRGs) are EGF-like ligands associated with cognitive disorders. Unprocessed proNRG3 is cleaved by BACE1 to generate the mature membrane-bound NRG3 ligand, but the subcellular site of proNRG3 cleavage, mechanisms underlying its transport into axons, and presynaptic accumulation remain unknown. Using an optogenetic proNRG3 cleavage reporter (LA143-NRG3), we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network but, upon photoactivation, is cleaved by BACE1 and released from the TGN. Mature NRG3 then emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via transcytosis. By contrast, the BACE1 substrate APP is sorted into axons on Rab11+ vesicles. 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., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

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. Herein we report the identification of the RUN- and FYVE-domain-containing proteins RUFY3 and RUFY4 as ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin for retrograde transport along microtubules. Using various methodologies, we find that RUFY3 and RUFY4 interact with both GTP-bound ARL8 and dynein-dynactin. In addition, we show that RUFY3 and RUFY4 promote concentration of endolysosomes in the juxtanuclear area of non-neuronal cells, and drive redistribution of endolysosomes from the axon to the soma in hippocampal neurons. The function of RUFY3 in retrograde transport contributes to the juxtanuclear redistribution of endolysosomes upon cytosol alkalinization. These studies thus identify RUFY3 and RUFY4 as ARL8-dependent, dynein-dynactin adaptors or regulators, and highlight the role of ARL8 in the control of both anterograde and retrograde endolysosome transport., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Lysosomes are membrane-bound organelles that degrade diverse biomolecules and regulate a multitude of other essential processes including cell growth and metabolism, signaling, plasma membrane repair and infection. Such diverse functions of lysosomes are highly coordinated in space and time and are therefore tightly coupled to the directional transport of the organelles within the cytoplasm. Thus, robust quantitative assessments of lysosome positioning within the cell provide a valuable tool for researchers interested in understanding these multifunctional organelles. Here, we present point-by-point methodology to measure lysosome positioning by two straight forward and widely used techniques: shell analysis and line scan., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show 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. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. 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., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Autophagy-related protein 9 (ATG9) is a transmembrane protein component of the autophagy machinery that cycles between the trans -Golgi network (TGN) in the perinuclear area and other compartments in the peripheral area of the cell. In mammalian cells, export of the ATG9A isoform from the TGN into ATG9A-containing vesicles is mediated by the adaptor protein 4 (AP-4) complex. However, the mechanisms responsible for the subsequent distribution of these vesicles to the cell periphery are unclear. Herein we show that the AP-4-accessory protein RUSC2 couples ATG9A-containing vesicles to the plus-end-directed microtubule motor kinesin-1 via an interaction between a disordered region of RUSC2 and the kinesin-1 light chain. This interaction is counteracted by the microtubule-associated protein WDR47. These findings uncover a mechanism for the peripheral distribution of ATG9A-containing vesicles involving the function of RUSC2 as a kinesin-1 adaptor and WDR47 as a negative regulator of this function.

Background: Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1)., Results: We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1 -/- i 3 Neuron) iPSC-derived neuron model of NPC1. The NPC1 -/- and the corresponding isogenic NPC1 +/+ i 3 Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1 -/- i 3 Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-β-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial., Conclusion: Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1 -/- i 3 Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration., (© 2021. The Author(s).)

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi 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. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin 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. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The accumulation of α-synuclein (α-syn) aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson disease (PD). α-Syn aggregates propagate in a "prion-like" manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs). However, how lysosomes participate in the spreading of α-syn aggregates is unclear. Here, by using super-resolution (SR) and electron microscopy (EM), we find that α-syn fibrils affect the morphology of lysosomes and impair their function in neuronal cells. In addition, we demonstrate that α-syn fibrils induce peripheral redistribution of lysosomes, likely mediated by transcription factor EB (TFEB), increasing the efficiency of α-syn fibrils' transfer to neighboring cells. We also show that lysosomal membrane permeabilization (LMP) allows the seeding of soluble α-syn in cells that have taken up α-syn fibrils from the culture medium, and, more importantly, in healthy cells in coculture, following lysosome-mediated transfer of the fibrils. Moreover, we demonstrate that seeding occurs mainly at lysosomes in both donor and acceptor cells, after uptake of α-syn fibrils from the medium and following their transfer, respectively. Finally, by using a heterotypic coculture system, we determine the origin and nature of the lysosomes transferred between cells, and we show that donor cells bearing α-syn fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them. These findings thus contribute to the elucidation of the mechanism by which α-syn fibrils spread through TNTs, while also revealing the crucial role of lysosomes, working as a Trojan horse for both seeding and propagation of disease pathology., Competing Interests: The authors declare no competing interests.

Long-range movement of organelles within the cytoplasm 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. Herein, we show that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small guanosine triphosphatase (GTPase) ARL8 on the lysosomal membrane to couple lysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP reveal that the C-terminal region comprising three pleckstrin homology (PH) domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of lysosomes to kinesin-1 and, consequently, lysosome movement toward the cell periphery. We also find that ARL8 does not just recruit SKIP to the lysosomal membrane but also relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde lysosome transport. Finally, our analyses show that the largely disordered middle region of SKIP mediates self-association and that this self-association enhances the interaction of SKIP with kinesin-1. These findings indicate that SKIP is not just a passive connector of lysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. We anticipate that similar organelle- or GTPase-induced conformational changes could regulate the activity of other kinesin adaptors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype., Competing Interests: The authors have declared that no competing interests exist.

ATG9, the only transmembrane protein in the core macroautophagy/autophagy machinery, is a key player in the early stages of autophagosome formation. Yet, the lack of a high-resolution structure of ATG9 was a major impediment in understanding its three-dimensional organization and function. We recently solved a high-resolution cryoEM structure of the ubiquitously expressed human ATG9A isoform. The structure revealed that ATG9A is a domain-swapped homotrimer with a unique fold, and has an internal network of branched cavities. In cellulo analyses demonstrated the functional importance of the cavity-lining residues. These cavities could serve as conduits for transport of hydrophilic moieties, such as lipid headgroups, across the bilayer. Finally, structure-guided molecular dynamics predicted that ATG9A has membrane-bending properties, which is consistent with its localization to highly curved membranes.

Heterotetrameric adaptor protein (AP) complexes play key roles in protein sorting and transport vesicle formation in the endomembrane system of eukaryotic cells. One of these complexes, AP-4, was identified over 20 years ago but, up until recently, its function remained unclear. AP-4 associates with the trans-Golgi network (TGN) through interaction with small GTPases of the ARF family and recognizes transmembrane proteins (i.e. cargos) having specific sorting signals in their cytosolic domains. Recent studies identified accessory proteins (tepsin, RUSC2 and the FHF complex) that co-operate with AP-4, and cargos (amyloid precursor protein, ATG9A and SERINC3/5) that are exported from the TGN in an AP-4-dependent manner. Defective export of ATG9A from the TGN in AP-4-deficient cells was shown to reduce ATG9A delivery to pre-autophagosomal structures, impairing autophagosome formation and/or maturation. In addition, mutations in AP-4-subunit genes were found to cause neurological dysfunction in mice and a form of complicated hereditary spastic paraplegia referred to as 'AP-4-deficiency syndrome' in humans. These findings demonstrated that mammalian AP-4 is required for the development and function of the central nervous system, possibly through its role in the sorting of ATG9A for the maintenance of autophagic homeostasis. In this article, we review the properties and functions of AP-4, and discuss how they might explain the clinical features of AP-4 deficiency., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Autophagy is a catabolic process involving capture of cytoplasmic materials into double-membraned autophagosomes that subsequently fuse with lysosomes for degradation of the materials by lysosomal hydrolases. One of the least understood components of the autophagy machinery is the transmembrane protein ATG9. Here, we report a cryoelectron microscopy structure of the human ATG9A isoform at 2.9-Å resolution. The structure reveals a fold with a homotrimeric domain-swapped architecture, multiple membrane spans, and a network of branched cavities, consistent with ATG9A being a membrane transporter. Mutational analyses support a role for the cavities in the function of ATG9A. In addition, structure-guided molecular simulations predict that ATG9A causes membrane bending, explaining the localization of this protein to small vesicles and highly curved edges of growing autophagosomes., Competing Interests: Declaration of Interests The authors declare no competing interest., (Published by Elsevier Inc.)

BORC is a multisubunit complex previously shown to promote coupling of mammalian lysosomes and C. elegans synaptic vesicle (SV) precursors (SVPs) to kinesins for anterograde transport of these organelles along microtubule tracks. We attempted to meld these observations into a unified model for axonal transport in mammalian neurons by testing two alternative hypotheses: (1) that SV and lysosomal proteins are co-transported within a single type of "lysosome-related vesicle" and (2) that SVPs and lysosomes are distinct organelles, but both depend on BORC for axonal transport. Analyses of various types of neurons from wild-type rats and mice, as well as from BORC-deficient mice, show that neither hypothesis is correct. We find that SVPs and lysosomes are transported separately, but only lysosomes depend on BORC for axonal transport in these neurons. These findings demonstrate that SVPs and lysosomes are distinct organelles that rely on different machineries for axonal transport in mammalian neurons., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Cell-to-cell transmission of misfolding-prone α-synuclein (α-Syn) has emerged as a key pathological event in Parkinson's disease. This process is initiated when α-Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and myosin-7B (MYO7B) as critical endocytosis regulators for α-Syn preformed fibrils (PFFs). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting α-Syn PFFs to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in α-Syn PFFs. By contrast, MYO7B regulates α-Syn PFF cell entry by maintaining a plasma membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing "scars" on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is restricted to α-Syn PFFs and other polycation-bearing cargos that enter cells via HSPGs. Thus, our study not only defines regulatory factors for α-Syn PFF endocytosis, but also reveals a previously unknown endocytosis mechanism for HSPG-binding cargos in general, which requires forces generated by MYO7B and actin filaments., Competing Interests: The authors declare no competing interest.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson's disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD., Competing Interests: Declaration of Interests M.J.F. is currently an employee of Merck Sharpe & Dohme Inc., (Published by Elsevier Inc.)

The heterotetrameric adaptor protein complex 4 (AP-4) is a component of a protein coat associated with the trans -Golgi network (TGN). Mutations in AP-4 subunits cause a complicated form of autosomal-recessive hereditary spastic paraplegia termed AP-4-deficiency syndrome. Recent studies showed that AP-4 mediates export of the transmembrane autophagy protein ATG9A from the TGN to preautophagosomal structures. To identify additional proteins that cooperate with AP-4 in ATG9A trafficking, we performed affinity purification-mass spectrometry followed by validation of the hits by biochemical and functional analyses. This approach resulted in the identification of the fused toes homolog-Hook-FHIP (FHF) complex as a novel AP-4 accessory factor. We found that the AP-4-FHF interaction is mediated by direct binding of the AP-4 μ4 subunit to coiled-coil domains in the Hook1 and Hook2 subunits of FHF. Knockdown of FHF subunits resulted in dispersal of AP-4 and ATG9A from the perinuclear region of the cell, consistent with the previously demonstrated role of the FHF complex in coupling organelles to the microtubule (MT) retrograde motor dynein-dynactin. These findings thus uncover an additional mechanism for the distribution of ATG9A within cells and provide further evidence for a role of protein coats in coupling transport vesicles to MT motors.

Exciting new discoveries have transformed the view of the lysosome from a static organelle dedicated to the disposal and recycling of cellular waste to a highly dynamic structure that mediates the adaptation of cell metabolism to environmental cues. Lysosome-mediated signalling pathways and transcription programmes are able to sense the status of cellular metabolism and control the switch between anabolism and catabolism by regulating lysosomal biogenesis and autophagy. The lysosome also extensively communicates with other cellular structures by exchanging content and information and by establishing membrane contact sites. It is now clear that lysosome positioning is a dynamically regulated process and a crucial determinant of lysosomal function. Finally, growing evidence indicates that the role of lysosomal dysfunction in human diseases goes beyond rare inherited diseases, such as lysosomal storage disorders, to include common neurodegenerative and metabolic diseases, as well as cancer. Together, these discoveries highlight the lysosome as a regulatory hub for cellular and organismal homeostasis, and an attractive therapeutic target for a broad variety of disease conditions.

Like other biological processes, macroautophagy/autophagy must be tightly controlled for maintenance of cellular homeostasis and for proper response to changing cellular conditions. To gain insights into the regulation of autophagy, we recently conducted a genome-wide CRISPR-Cas9 knockout screen using cells expressing endogenous LC3B tagged with GFP-mCherry as a reporter. This approach allowed us to identify the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B, enhancing the degradation of autophagy adaptors and the clearance of intracellular proteins aggregates. This finding could be the basis for the development of pharmacological inhibitors of UBA6 or BIRC6 for the treatment of protein aggregation disorders. Recent work by another group showed that BIRC6 itself is subject to ubiquitination and proteasomal degradation, highlighting the existence of a complex regulatory network for the control of LC3B levels.

Although the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. To identify novel autophagy regulators, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroglioma cells expressing endogenous LC3B tagged with a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as autophagy regulators. We found that these enzymes cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increased autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, UBA6 or BIRC6 depletion decreased the formation of aggresome-like induced structures in H4 cells, and α-synuclein aggregates in rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B. Inhibition of UBA6/BIRC6 could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders., Competing Interests: RJ, JB No competing interests declared

SNARE-mediated fusion of endosome-derived transport carriers with the trans-Golgi network (TGN) depends on the concerted action of two types of tethering factors: long coiled-coil tethers of the golgin family, and the heterotetrameric complex GARP. Whereas the golgins mediate long-distance capture of the carriers, GARP promotes assembly of the SNAREs. It remains to be determined, however, how the functions of these tethering factors are coordinated. Herein we report that the ARF-like (ARL) GTPase ARFRP1 functions upstream of two other ARL GTPases, ARL1 and ARL5, which in turn recruit golgins and GARP, respectively, to the TGN. We also show that this mechanism is essential for the delivery of retrograde cargos to the TGN. Our findings thus demonstrate that ARFRP1 is a master regulator of retrograde-carrier tethering to the TGN. The coordinated recruitment of distinct tethering factors by a bifurcated GTPase cascade may be paradigmatic of other vesicular fusion events within the cell., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)