Endosomal Recycling Research Articles

In the fast lane: Receptor trafficking during status epilepticus.

Status epilepticus (SE) remains a significant cause of morbidity and mortality and often is refractory to standard first-line treatments. A rapid loss of synaptic inhibition and development of pharmacoresistance to benzodiazepines (BZDs) occurs early during SE, while NMDA and AMPA receptor antagonists remain effective treatments after BZDs have failed. Multimodal and subunit-selective receptor trafficking within minutes to an hour of SE involves GABA-A, NMDA, and AMPA receptors and contributes to shifts in the number and subunit composition of surface receptors with differential impacts on the physiology, pharmacology, and strength of GABAergic and glutamatergic currents at synaptic and extrasynaptic sites. During the first hour of SE, synaptic GABA-A receptors containing γ2 subunits move to the cell interior while extrasynaptic GABA-A receptors with δ subunits are preserved. Conversely, NMDA receptors containing N2B subunits are increased at synaptic and extrasynaptic sites, and homomeric GluA1 ("GluA2-lacking") calcium permeant AMPA receptor surface expression also is increased. Molecular mechanisms, largely driven by NMDA receptor or calcium permeant AMPA receptor activation early during circuit hyperactivity, regulate subunit-specific interactions with proteins involved with synaptic scaffolding, adaptin-AP2/clathrin-dependent endocytosis, endoplasmic reticulum (ER) retention, and endosomal recycling. Reviewed here is how SE-induced shifts in receptor subunit composition and surface representation increase the excitatory to inhibitory imbalance that sustains seizures and fuels excitotoxicity contributing to chronic sequela such as "spontaneous recurrent seizures" (SRS). A role for early multimodal therapy is suggested both for treatment of SE and for prevention of long-term comorbidities.

STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

Accessory ESCRT-III proteins are conserved and selective regulators of Rab11a-exosome formation.

Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7-positive multivesicular endosomes, and also in recycling Rab11a-positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos. Accessory ESCRT-III components have reported roles in ESCRT-III-mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT-III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a-enriched exosome preparations. We show that these proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a-exosome production. Accessory ESCRT-III knockdown suppresses seminal fluid-mediated reproductive signalling by secondary cells and the growth-promoting activity of Rab11a-exosome-containing EVs from HCT116 cells. We conclude that accessory ESCRT-III components have a specific, ubiquitin-independent role in Rab11a-exosome generation, a mechanism that might be targeted to selectively block pro-tumorigenic activities of these vesicles in cancer.

Exploring RAB11A Pathway to Hinder Chronic Myeloid Leukemia-Induced Angiogenesis In Vivo.

Neoangiogenesis is generally correlated with poor prognosis, due to the promotion of cancer cell growth, invasion and metastasis. The progression of chronic myeloid leukemia (CML) is frequently associated with an increased vascular density in bone marrow. From a molecular point of view, the small GTP-binding protein Rab11a, involved in the endosomal slow recycling pathway, has been shown to play a crucial role for the neoangiogenic process at the bone marrow of CML patients, by controlling the secretion of exosomes by CML cells, and by regulating the recycling of vascular endothelial factor receptors. The angiogenic potential of exosomes secreted by the CML cell line K562 has been previously observed using the chorioallantoic membrane (CAM) model. Herein, gold nanoparticles (AuNPs) were functionalized with an anti-RAB11A oligonucleotide (AuNP@RAB11A) to downregulate RAB11A mRNA in K562 cell line which showed a 40% silencing of the mRNA after 6 h and 14% silencing of the protein after 12 h. Then, using the in vivo CAM model, these exosomes secreted by AuNP@RAB11A incubated K562 did not present the angiogenic potential of those secreted from untreated K562 cells. These results demonstrate the relevance of Rab11 for the neoangiogenesis mediated by tumor exosomes, whose deleterious effect may be counteracted via targeted silencing of these crucial genes; thus, decreasing the number of pro-tumoral exosomes at the tumor microenvironment.

Restoration of β-adrenergic responsivity in the aging heart.

Progressive cardiac hypo-responsivity to β-adrenergic receptor (βAR)-stimulation occurs with aging, diminishing capacity to manage acute stress, and rendering the heart more vulnerable to systemic stressors including hypertension and diabetes. Disruption of this crucial regulatory mechanism reduces inotropic reserve and lowers the threshold for heart failure. Yet the causal links between aging and β-AR hypo-responsivity are unclear. Here, using electrophysiology, advanced imaging approaches and biochemistry, we propose a mechanism that explains this age-dependent hypo-responsivity, and a restorative measure to rejuvenate the aging heart. Our results indicate that cardiac Ca2+ channels CaV1.2 and type 2 ryanodine receptors display altered nanoscale distribution, and impaired βAR-stimulated reorganization and recycling with aging that limits their functional augmentation. We find that age-associated overexpression of bridging integrator 1 (BIN1) contributes to dysregulated endosomal recycling and disrupted trafficking dynamics of these channels. BIN1 knockdown restored βAR-signaling responsivity and inotropic reserve to youthful levels. Paired echocardiograms of old mice before and after BIN1 knockdown revealed a restoration in cardiac contractility in vivo. In conclusion, we find that βAR hypo-responsivity in the aging heart occurs in part due to expressional upregulation of BIN1 that contributes to deficits in the endosomal recycling pathway, resulting in enlarged endosomes, altered trafficking dynamics of cardiac Ca2+ channels, and limiting the plasticity of excitation-contraction coupling. We propose targeted BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

BIN1 is essential for β-adrenergic regulation of cardiac function.

The number and activity of voltage gated L-type CaV1.2 Ca2+ channels in the sarcolemma of cardiomyocytes tunes the magnitude of Ca2+-induced Ca2+ release and subsequent contraction. Previous studies from our group revealed that β-adrenergic receptor stimulation in mouse ventricular myocytes triggers mobilization of CaV1.2 channels from early and recycling endosomal reservoirs to augment sarcolemmal expression and clustering and enhancing excitation-contraction coupling. In the heart, the membrane curvature-mediating protein bridging integrator 1 (BIN1) mediates t-tubule micro-folding, and targeted delivery of CaV1.2 channels to t-tubules via anchored microtubules. BIN1 deficits are associated with heart failure. In neurons, BIN1 is known to play an essential role in tubulation of endosomes to permit cargo exit and recycling. We used myocytes isolated from cardiac-specific BIN1 knockout mice, to test the hypothesis that expressional deficits in BIN1 impairs isoproterenol-stimulated endosomal recycling of CaV1.2, reducing the ability to respond to acute stress. Super-resolution Airyscan imaging of immunostained ventricular myocytes revealed that CaV1.2 pools on early and recycling endosomes are mobilized to the sarcolemma to bolster channel expression in response to β-adrenergic stimulation with isoproterenol in WT but not BIN1-/- cells. Moreover, single molecule localization microscopy revealed that isoproterenol-stimulated CaV1.2 upregulation and super-clustering at the t-tubule sarcolemma occurs in wild-type myocytes but is reduced in BIN1+/- and absent in BIN1-/- myocytes. To assess the functional implications on β-adrenergic regulation of CaV1.2 and excitation-contraction coupling, we performed voltage-clamp electrophysiological recordings of whole-cell Ca2+ currents and measured Ca2+transients from paced myocytes finding impaired responses to isoproterenol in BIN1 knockout cells versus wild-type controls. Based on these data, we propose that tight regulation of BIN1 expression is required to support β-adrenergic receptor stimulated CaV1.2 channel trafficking and recycling to increase Ca2+ influx and inotropy in times of acute stress.

Eukaryotic Clathrin Adapter Protein and Mediator of Cholesterol Homeostasis, PICALM, Affects Trafficking to the Chlamydial Inclusion.

The obligate intracellular pathogen Chlamydia trachomatis has unique metabolic requirements as it proceeds through its biphasic developmental cycle from within the inclusion within the host cell. In our previous study, we identified a host protein, PICALM, which localizes to the chlamydial inclusion. PICALM functions in many host pathways including the recycling of receptors, specific SNARE proteins, and molecules like transferrin, and maintaining cholesterol homeostasis. Hence, we hypothesized that PICALM functions to maintain the cholesterol content and to moderate trafficking from the endosomal recycling pathway to the inclusion, which controls chlamydial access to this pathway. In uninfected cells, siRNA knockdown of PICALM resulted in increased cholesterol within the Golgi and transferrin receptor (TfR) positive vesicles (recycling endosomes). PICALM knockdown in cells infected with C. trachomatis resulted in increased levels of Golgi-derived lipid and protein, TfR, transferrin, and Rab11-FIP1 localized to inclusions and a decrease of Golgi fragmentation at and Rab11 trafficking to the inclusion. Interestingly, chlamydial infection alone also increases cholesterol in TfR and Rab11-associated vesicles, and PICALM knockdown reverses this effect. Our data suggest that PICALM functions to balance or limit chlamydial access to multiple subcellular trafficking pathways to maintain the health of the host cell during chlamydial infection.

Dimerization of the Alzheimer’s disease pathogenic receptor SORLA regulates its association with retromer

SORL1, the gene encoding the large multidomain SORLA protein, has emerged as only the fourth gene that when mutated can by itself cause Alzheimer's disease (AD), and as a gene reliably linked to both the early- and late-onset forms of the disease. SORLA is known to interact with the endosomal trafficking regulatory complex called retromer in regulating the recycling of endosomal cargo, including the amyloid precursor protein (APP) and the glutamate receptor GluA1. Nevertheless, SORLA's precise structural-functional relationship in endosomal recycling tubules remains unknown. Here, we address these outstanding questions by relying on crystallographic and artificial-intelligence evidence to generate a structural model for how SORLA folds and fits into retromer-positive endosomal tubules, where it is found to dimerize via both SORLA's fibronectin-type-III (3Fn)- and VPS10p-domains. Moreover, we identify a SORLA fragment comprising the 3Fn-, transmembrane, and cytoplasmic domains that has the capacity to form a dimer, and to enhance retromer-dependent recycling of APP by decreasing its amyloidogenic processing. Collectively, these observations generate a model for how SORLA dimer (and possibly polymer) formation can function in stabilizing and enhancing retromer function at endosome tubules. These findings can inform investigation of the many AD-associated SORL1 variants for evidence of pathogenicity and can guide discovery of novel drugs for the disease.

Elevated transferrin receptor impairs T cell metabolism and function in systemic lupus erythematosus.

T cells in systemic lupus erythematosus (SLE) exhibit multiple metabolic abnormalities. Excess iron can impair mitochondria and may contribute to SLE. To gain insights into this potential role of iron in SLE, we performed a CRISPR screen of iron handling genes on T cells. Transferrin receptor (CD71) was identified as differentially critical for TH1 and inhibitory for induced regulatory T cells (iTregs). Activated T cells induced CD71 and iron uptake, which was exaggerated in SLE-prone T cells. Cell surface CD71 was enhanced in SLE-prone T cells by increased endosomal recycling. Blocking CD71 reduced intracellular iron and mTORC1 signaling, which inhibited TH1 and TH17 cells yet enhanced iTregs. In vivo treatment reduced kidney pathology and increased CD4 T cell production of IL-10 in SLE-prone mice. Disease severity correlated with CD71 expression on TH17 cells from patients with SLE, and blocking CD71 in vitro enhanced IL-10 secretion. T cell iron uptake via CD71 thus contributes to T cell dysfunction and can be targeted to limit SLE-associated pathology.

A Bispecific METxMET Antibody-Drug Conjugate with Cleavable Linker Is Processed in Recycling and Late Endosomes.

Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.

Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV uses a physiological liver-directed pathway that supports specific host-cell targeting invivo. We established the exvivo perfusion of intact human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the invivo situation. HBV was rapidly sequestered by liver macrophages within 1 hour after a virus pulse perfusion but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins in serum and within machrophages. Electron and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilize the hepatocyte-directed cholesterol transport machinery of macrophages. Our results propose that by binding to liver targeted lipoproteins and using the reverse cholesterol transport pathway of macrophages, HBV hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve transinfection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.

The trans‐entorhinal cortex differential depends on retromer‐mediated endosomal recycling

AbstractAnatomical studies have pinpointed the trans‐entorhinal cortex (TEC) as the subregion of the brain selectively vulnerable to Alzheimer’s disease. Additional molecular studies have identified endosomal trafficking defects as a cytopathological driver of the disease. However, whether and how endosomal trafficking is linked to anatomical vulnerability still remains unknown. Here, in addressing these questions, we begin by showing that neurons are enriched with a functional distinct retromer core organized around VPS26b, differentially dedicated to endosomal recycling. Using fMRI imaging, and in contrast to its paralog VPS26a, we further demonstrated that depleting VPS26b in mice caused preferential age‐dependent dysfunction in the trans‐entorhinal cortex, a finding further validated by electrophysiology, immunocytochemistry, and behavior. Surprisingly, repletion of VPS26b, both in vitro and in vivo, rescued all phenotypes seen upon VPS26b depletion. We then turned to humans to find VPS26b highly enriched in the TEC region, where both VPS26b and the retromer‐related receptor SORL1 were found deficient in Alzheimer’s brains. Finally, by regulating glutamate receptor and SORL1 recycling and its levels, VPS26b was shown to mediate regionally‐selective synaptic dysfunction, as well as AD‐associated amyloid‐precursor protein misprocessing and tau secretion. Together with the trans‐entorhinal’s unique network properties, hypothesized to impose a heavy demand on endosomal recycling, these results suggest a general mechanism that can explain AD’s regional vulnerability.

SORL1‐retromer regulates endosomal recycling and helps explain Alzheimer’s regional vulnerability

AbstractThe importance of the endosomal recycling pathway for cellular health was recognized ever since its reverse trafficking itinerary, clathrin‐dependent endocytosis, was described. Vital for synaptic function, a recent study found that neurons are endowed with a distinct unit dedicated to the recycling pathway. Akin to how endocytosis is regulated by a functional unit formed by the clathrin coat interacting with low‐density lipoprotein‐related receptors (LDLRs), recent studies will be reviewed suggesting that endosomal recycling in neurons is regulated by a functional unit comprised of a neuronal‐enriched retromer coat that interacts with the sortilin‐related receptor SORL1. Genetic studies suggest that rare truncating mutations in SORL1, inducing SORL1 haploinsuffiency, acts a causal event in triggering Alzheimer’s disease (AD). In agreement with this interpretation, recent studies will be reviewed showing that SORL1 haploinsufficiency can trigger AD’s cardinal pathologies, including showing in mice that it leads to selective atrophy in the trans‐entorhinal cortex. These studies suggest that SORL1‐retromer recycling is a causal pathway in AD that when disrupted can trigger AD’s molecular, cellular, and anatomical pathologies. When interpreted together with a collection of other studies this recent insight suggests why the trans‐entohrinal cortex is so dependent on SORL1‐retromer recycling, why the trans‐entorhinal cortex is differentially vulnerable even to ‘sporadic’ AD, and why AD anatomically ‘spreads’ over time.

Endosomal Localization of RacGAP Protein ARHGAP22 Regulates its GAP Activity in Human Melanoma Cells.

Rho small GTPases regulate cancer cell adhesion, migration and invasion through reorganization of the actin cytoskeleton. Rho GTPase Activating Protein 22 (ARHGAP22) is a Rac-specific GAP and suppresses Rac-dependent lamella formation and cell spreading. We have previously shown that ARHGAP22 localizes at endosomes in human melanoma A7 cells. The aim of the present study was to demonstrate the functional significance of its localization at the endosomes in melanoma cells. The lamella formation and cell spreading were monitored using human melanoma A7 cells. The effect of inhibition of endosome recycling pathway was examined. We found that dominant negative Rab11 S25N mutant inhibits RacGAP activity of ARHGAP22 and blocks ARHGAP22-dependent suppression of lamella formation and melanoma cell spreading. Furthermore, deletion of 19 amino acid residues at the C-terminal region of ARHGAP22 abolished the localization of ARHGAP22 at enlarged vesicles and stimulated RacGAP activity of ARHGAP22. The deletion mutant accumulated at enlarged vesicles when endosome recycling pathway was blocked either by co-transfection of the Rab11 S25N mutant or treatment of the cells with N-ethylmaleimide, which blocks endosomal vesicular fusion to the plasma membrane. On the other hand, deletion of the pleckstrin homology (PH) domain of ARHGAP22 abolished its RacGAP activity and localization at the plasma membrane. ARHGAP22 localizes at endosomes and is transported to the plasma membrane to inactivate Rac and suppresses lamella formation and spreading of melanoma cells.

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

Dynamic regulation of KIF15 phosphorylation and acetylation promotes focal adhesions disassembly in pancreatic cancer

Pancreatic cancer (PC) is prone to distant metastasis in the early stage, which is attributed to the strong migration ability of tumor cells. Focal adhesion turnover is essential for cancer cell metastasis, and the integrin recycling process is a key activation pathway for focal adhesion depolymerization. To identify the key motor protein involving in the integrin β1 recycling, we screened kinesin proteins involved in integrin β1 recycling using a kinesin family siRNA library and identified kinesin family 15 (KIF15) as a key regulator. KIF15 was upregulated in metastasis PC tissues and promoted PC cell migration and invasion. We identified KIF15 as a key component mediating integrin β1/FAK signaling that accelerated FA disassembly in a FAK-Y397-dependent manner. KIF15 recruited PI3K-C2α to promote integrin β1/FAK signaling and FA disassembly in a RAB11A-dependent manner. The C-terminal tail of KIF15 is required for the PI3K-C2α interaction and RAB11A activation. In addition, we also found that SIRT1-mediated acetylation of KIF15 is essential for KIF15 phosphorylation, which is the key activation event in motor protein function. Together, these findings indicate that KIF15 interacts with PI3K-C2α to promote FA turnover in PC cells by controlling the endosome recycling of integrin β1 in a SIRT1 acetylation modification-dependent manner, eventually promoting focal adhesions turnover and distant metastasis in PC.

WASH activation controls endosomal recycling and EGFR and Hippo signaling during tumor-suppressive cell competition

Cell competition is a conserved homeostatic mechanism whereby epithelial cells eliminate neighbors with lower fitness. Cell communication at the interface of wild-type “winner” cells and polarity-deficient (scrib−/−) “losers” is established through Sas-mediated Ptp10D activation in polarity-deficient cells. This tumor-suppressive cell competition restrains EGFR and Hippo signaling and enables Eiger-JNK mediated apoptosis in scrib−/− clones. Here, we show that the activation state of the endosomal actin regulator WASH is a central node linking EGFR and Hippo signaling activation. The tyrosine kinase Btk29A and its substrate WASH are required downstream of Ptp10D for “loser” cell elimination. Constitutively active, phosphomimetic WASH is sufficient to induce both EGFR and Yki activation leading to overgrowth. On the mechanistic level we show that Ptp10D is recycled by the WASH/retromer complex, while EGFR is recycled by the WASH/retriever complex. Constitutive WASH activation selectively interferes with retromer function leading to Ptp10D mistargeting while promoting EGFR recycling and signaling activation. Phospho-WASH also activates aberrant Arp2/3 actin polymerization, leading to cytoskeletal imbalance, Yki activation and reduced apoptosis. Selective manipulation of WASH phosphorylation on sorting endosomes may restrict epithelial tumorous growth.

A second chance at yeast early endosomes.

Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109137) describe a yeast endosomal recycling pathway that gives proteins a second chance to return to the plasma membrane.

Convergence of secretory, endosomal, and autophagic routes in trans-Golgi-associated lysosomes.

At the trans-Golgi, complex traffic connections exist to the endolysosomal system additional to the main Golgi-to-plasma membrane secretory route. Here, we investigated three hits in a Drosophila screen displaying secretory cargo accumulation in autophagic vesicles: ESCRT-III component Vps20, SNARE-binding Rop, and lysosomal pump subunit VhaPPA1-1. We found that Vps20, Rop, and lysosomal markers localize near the trans-Golgi. Furthermore, we document that the vicinity of the trans-Golgi is the main cellular location for lysosomes and that early, late, and recycling endosomes associate as well with a trans-Golgi-associated degradative compartment where basal microautophagy of secretory cargo and other materials occurs. Disruption of this compartment causes cargo accumulation in our hits, including Munc18 homolog Rop, required with Syx1 and Syx4 for Rab11-mediated endosomal recycling. Finally, besides basal microautophagy, we show that the trans-Golgi-associated degradative compartment contributes to the growth of autophagic vesicles in developmental and starvation-induced macroautophagy. Our results argue that the fly trans-Golgi is the gravitational center of the whole endomembrane system.

WDR91 specifies the endosomal retrieval subdomain for retromer-dependent recycling.

Retromer-dependent endosomal recycling of membrane receptors requires Rab7, sorting nexin (SNX)-retromer, and factors that regulate endosomal actin organization. It is not fully understood how these factors cooperate to form endosomal subdomains for cargo retrieval and recycling. Here, we report that WDR91, a Rab7 effector, is the key factor that specifies the endosomal retrieval subdomain. Loss of WDR91 causes defective recycling of both intracellular and cell surface receptors. WDR91 interacts with SNXs through their PX domain, and with VPS35, thus promoting their interaction with Rab7. WDR91 also interacts with the WASH subunit FAM21. In WDR91-deficient cells, Rab7, SNX-retromer, and FAM21 fail to localize to endosomal subdomains, and endosomal actin organization is impaired. Re-expression of WDR91 enables Rab7, SNX-retromer, and FAM21 to concentrate at WDR91-specific endosomal subdomains, where retromer-mediated membrane tubulation and release occur. Thus, WDR91 coordinates Rab7 with SNX-retromer and WASH to establish the endosomal retrieval subdomains required for retromer-mediated endosomal recycling.