Pre-autophagosomal Structure Research Articles

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata

BackgroundBiofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach.ResultsFunctional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements.ConclusionsThe study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

Isobavachalcone exhibits antifungal and antibiofilm effects against C. albicans by disrupting cell wall/membrane integrity and inducing apoptosis and autophagy.

Isobavachalcone (IBC) is a natural flavonoid with multiple pharmacological properties. This study aimed to evaluate the efficacy of IBC against planktonic growth and biofilms of Candida albicans (C. albicans) and the mechanisms underlying its antifungal action. The cell membrane integrity, cell metabolic viability, and cell morphology of C. albicans treated with IBC were evaluated using CLSM and FESEM analyses. Crystal violet staining, CLSM, and FESEM were used to assess the inhibition of biofilm formation, as well as dispersal and killing effects of IBC on mature biofilms. RNA-seq combined with apoptosis and autophagy assays was used to examine the mechanisms underlying the antifungal action of IBC. IBC exhibited excellent antifungal activity with 8 μg/mL of MIC for C. albicans. IBC disrupted the cell membrane integrity, and inhibited biofilm formation. IBC dispersed mature biofilms and damaged biofilm cells of C. albicans at 32 μg/mL. Moreover, IBC induced apoptosis and autophagy-associated cell death of C. albicans. The RNA-seq analysis revealed upregulation or downregulation of key genes involved in cell wall synthesis (Wsc1 and Fks1), ergosterol biosynthesis (Erg3, and Erg11), apoptisis (Hsp90 and Aif1), as well as autophagy pathways (Atg8, Atg13, and Atg17), and so forth, in response to IBC, as evidenced by the experiment-based phenotypic analysis. These results suggest that IBC inhibits C. albicans growth by disrupting the cell wall/membrane, caused by the altered expression of genes associated with β-1,3-glucan and ergosterol biosynthesis. IBC induces apoptosis and autophagy-associated cell death by upregulating the expression of Hsp90, and altering autophagy-related genes involved in the formation of the Atg1 complex and the pre-autophagosomal structure. Together, our findings provide important insights into the potential multifunctional mechanism of action of IBC.

A novel fluorescence-activated cell sorting (FACS)-based screening identified ATG14, the gene required for pexophagy in the methylotrophic yeast.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Association and Functional Analysis of Ferroptosis-related Genes with Prostate Hyperplasia

Objective: This study aimed to investigate the association and biological functions of ferroptosis-related genes with prostatic hyperplasia. Methods: Gene information from ferroptosis and prostatic hyperplasia databases was used to screen for ferroptosis-related genes associated with prostate hyperplasia. Protein-protein interaction (PPI), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and enrichment analysis were employed to evaluate the interaction between these genes and prostatic hyperplasia. Results: Through PPI, GO, KEGG, and enrichment analysis, several ferroptosis-related genes associated with prostatic hyperplasia were identified, including phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), mitogen-activated protein kinase 3 (MAPK3), GABA type A receptor associated protein like 1 (GABARAPL1), HRas proto-oncogene (HRAS), tumor suppressor gene p53 (TP53), MAPK1, and autophagy related protein 7 (ATG7). These genes were found to be involved in various biological processes and signaling pathways related to ubiquitin protein ligase binding, protein phosphatase binding, pre-autophagosomal structure, autophagy, the PI3K-Akt signaling pathway and so on. Core genes such as PIK3CA, MAPK3, GABARAPL1, HRAS, TP53, MAPK1, ATG7, EGFR, Kirsten rat sarcoma viral oncogene homolog, and coiled-coil myosin-like BCL2-interacting protein were also found to be associated with transcription factors MYC, ESR2, PML, JUN, and TP53. Protein drug prediction suggested that FTI-277, Ginkgolide A, Tipifarnib, Desvenlafaxine, and Defactinib could potentially target the core genes. Conclusion: This study revealed the association and biological functions of ferroptosis-related genes with prostatic hyperplasia, providing new clues and directions for understanding the pathophysiology of prostatic hyperplasia and selecting therapeutic targets. These findings also contributed to a deeper understanding of the relationship between iron metabolism and prostatic hyperplasia, providing new ideas and methods for the diagnosis and treatment of iron metabolism-related diseases.

A GCC repeat in RAB26 undergoes natural selection in human and harbors divergent genotypes in late-onset Alzheimer’s disease

Although mainly located in genic regions and being mutation hotspots, intact blocks of CG-rich trinucleotide short tandem repeats (STRs) are largely overlooked with respect to their link with natural selection. The human RAB26 (member RAS oncogene family) directs synaptic and secretory vesicles into preautophagosomal structures, inhibition of which specifically disrupts axonal transport of degradative organelles and leads to an axonal dystrophy, resembling Alzheimer's disease (AD). Human RAB26 contains a GCC repeat in the top 1st percent in respect of length. Here we sequenced this STR in 441 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 216) and controls (N = 225). In both groups, the 12-repeat allele and the 12/12 genotype were predominantly abundant. We found excess of homozygosity for non-12 alleles in the NCD group (Mid-P exact = 0.027). Furthermore, divergent genotypes were detected that were specific to the NCD group (2.8% of genotypes) (Mid-P exact = 0.006) or controls (3.1% of genotypes) (Mid-P exact = 0.004). The patients harboring divergent genotypes received the diagnosis of AD. Based on the predominant abundance of the 12-repeat and 12/12 genotype in both groups, excess of non-12 homozygosity in the NCD group, and divergent genotypes across the NCD and control groups, we propose natural selection at this locus and link with late-onset AD. Our findings strengthen the hypothesis that a collection of rare genotypes unambiguously contribute to the pathogenesis of late-onset NCDs, such as AD.

The Atg1 complex, Atg9, and Vac8 recruit PI3K complex I to the pre-autophagosomal structure.

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14-Vac8 interaction is constitutive, the Atg38-Atg1 complex interaction and the Vps30-Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Pexophagy suppresses ROS-induced damage in leaf cells under high-intensity light

Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.

Atg1 kinase activity links PAS dissolution to balanced Atg8 conjugation.

Atg1 phosphoregulates different steps and factors in autophagy. Schreiber et al. report in Molecular Cell on the cell-free identification of a negative feedback ejection of Atg1 from the pre-autophagosomal structure (PAS), followed by positive feedback recruitment of Atg1 to phagophore-resident Atg8-PE, followed by yet another, negative feedback inhibition of the Atg8 conjugation machinery.

MoOpy2 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae.

The development and pathogenicity of the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae. However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae. Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the ΔMoopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation

SummaryAutophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and invitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

A conserved ATG2 binding site in WIPI4 and yeast Hsv2 is disrupted by mutations causing β-propeller protein-associated neurodegeneration.

PROPPINs are phosphoinositide-binding β-propeller proteins that mediate membrane recruitment of other proteins and are involved in different membrane remodeling processes. The main role of PROPPINs is their function in autophagy, where they act at different steps in phagophore formation. The human PROPPIN WIPI4 (WDR45) forms a complex with ATG2 involved in phagophore elongation, and mutations in this gene cause β-propeller protein-associated neurodegeneration (BPAN). The yeast functional counterpart of WIPI4 is Atg18, although its closest sequence homolog is another member of the PROPPIN family, Hsv2, whose function remains largely undefined. Here, we provide evidence that Hsv2, like WIPI4 and Atg18, interacts with Atg2. We show that Hsv2 and a pool of Atg2 colocalize on endosomes under basal conditions and at the pre-autophagosomal structure (PAS) upon autophagy induction. We further show that Hsv2 drives the recruitment of Atg2 to endosomes while Atg2 mediates Hsv2 recruitment to the PAS. HSV2 overexpression results in mis-sorting and secretion of carboxypeptidase CPY, suggesting that the endosomal function of this protein is related to the endosome-to-Golgi recycling pathway. Furthermore, we show that the Atg2 binding site is conserved in Hsv2 and WIPI4 but not in Atg18. Notably, two WIPI4 residues involved in ATG2 binding are mutated in patients with BPAN, and there is a correlation between the inhibitory effect of these mutations on ATG2 binding and the severity of the disease.

ATG16L1 functions in cell homeostasis beyond autophagy

Atg16-like (ATG16L) proteins were identified in higher eukaryotes for their resemblance to Atg16, a yeast protein previously characterized as a subunit of the Atg12-Atg5/Atg16 complex. In yeast, this complex catalyzes the lipidation of Atg8 on pre-autophagosomal structures and is therefore required for the formation of autophagosomes. In higher eukaryotes, ATG16L1 is also almost exclusively present as part of an ATG12-ATG5/ATG16L1 complex and has the same essential function in autophagy. However, ATG16L1 is three times bigger than Atg16. It displays, in particular, a carboxy-terminal extension, including a WD40 domain, which provides a platform for interaction with a variety of proteins, and allows for the recruitment of the ATG12-ATG5/ATG16L1 complex to membranes under different contexts. Furthermore, detailed analyses at the cellular level have revealed that some of the ATG16L1-driven activities are independent of the lipidation reaction catalyzed by the ATG12-ATG5/ATG16L1 complex. At the organ level, the use of mice that are hypomorphic for Atg16l1, or with cell-specific ablation of its expression, revealed a large panel of consequences of ATG16L1 dysfunctions. In this review, we recapitulate the current knowledge on ATG16L1 expression and functions. We emphasize, in particular, how it broadly acts as a brake on inflammation, thereby contributing to maintaining cell homeostasis. We also report on independent studies that converge to show that ATG16L1 is an important player in the regulation of intracellular traffic. Overall, autophagy-independent functions of ATG16L1 probably account for more of the phenotypes associated with ATG16L1 deficiencies than currently appreciated.

Atg27p co-fractionates with clathrin-coated vesicles in budding yeast.

Atg27p, a single-pass transmembrane protein that functions in autophagy, localizes to a variety of cellular compartments including the pre-autophagosomal structure, late Golgi, vacuolar membrane, as well as early and late endosomes. Its cytoplasmic C-terminus contains a tyrosine sorting motif that allows for its transport to the vacuolar membrane and an additional sequence that allows for its retrieval from the vacuolar membrane to the endosome. Since clathrin is well known to mediate vesicular transport in the endomembrane system, the trafficking of Atg27p and its tyrosine sorting motif suggested that it might be trafficked inside clathrin-coated vesicles (CCVs). In our previous studies, Atg27p was identified by mass spectrometry as a potential component in CCVs, as it was present in CCVs isolated from both WT and auxilin-depleted cells. We now confirm that Atg27p is a component of CCVs using immunoblotting and additional mass spectrometry data.

Atg27p localization is clathrin- and Ent3p/5p-dependent.

The autophagy-related protein Atg27p has been previously shown to localize to the autophagy-specific pre-autophagosomal structure (PAS) as well as to several organelles, including the late Golgi, the vacuolar membrane, and the endosome. Given that Atg27p localization to the vacuolar membrane in particular has been shown to be dependent on both its C-terminal tyrosine sorting motif and the AP-3 adaptor, and that Atg27p can be found in clathrin-coated vesicles, we set out to determine whether Atg27p localization inside cells is dependent on clathrin or on any of its cargo adaptors. We report that Atg27p localization is clathrin- and Ent3p/5p-dependent.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

Hepatitis B virus X Protein Promotes Liver Cancer Progression through Autophagy Induction in Response to TLR4 Stimulation.

Hepatitis B virus X (HBx) protein has been reported as a key protein regulating the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). Recent evidence has shown that HBx is implicated in the activation of autophagy in hepatic cells. Nevertheless, the precise molecular and cellular mechanism by which HBx induces autophagy is still controversial. Herein, we investigated the molecular and cellular mechanism by which HBx is involved in the TRAF6-BECN1-Bcl-2 signaling for the regulation of autophagy in response to TLR4 stimulation, therefore influencing the HCC progression. HBx interacts with BECN1 (Beclin 1) and inhibits the association of the BECN1-Bcl-2 complex, which is known to prevent the assembly of the pre-autophagosomal structure. Furthermore, HBx enhances the interaction between VPS34 and TRAF6-BECN1 complex, increases the ubiquitination of BECN1, and subsequently enhances autophagy induction in response to LPS stimulation. To verify the functional role of HBx in liver cancer progression, we utilized different HCC cell lines, HepG2, SK-Hep-1, and SNU-761. HBx-expressing HepG2 cells exhibited enhanced cell migration, invasion, and cell mobility in response to LPS stimulation compared to those of control HepG2 cells. These results were consistently observed in HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells. Taken together, our findings suggest that HBx positively regulates the induction of autophagy through the inhibition of the BECN1-Bcl-2 complex and enhancement of the TRAF6-BECN1-VPS34 complex, leading to enhance liver cancer migration and invasion.

The role of AP-4 in cargo export from the trans-Golgi network and hereditary spastic paraplegia.

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.

The FTS-Hook-FHIP (FHF) complex interacts with AP-4 to mediate perinuclear distribution of AP-4 and its cargo ATG9A.

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.