Selective Autophagy Pathway Research Articles

Induced Pluripotent Stem Cell Neuronal Models for the Study of Autophagy Pathways in Human Neurodegenerative Disease.

Human induced pluripotent stem cells (hiPSCs) are invaluable tools for research into the causes of diverse human diseases, and have enormous potential in the emerging field of regenerative medicine. Our ability to reprogramme patient cells to become hiPSCs, and to subsequently direct their differentiation towards those classes of neurons that are vulnerable to stress, is revealing how genetic mutations cause changes at the molecular level that drive the complex pathogeneses of human neurodegenerative diseases. Autophagy dysregulation is considered to be a major contributor in neural decline during the onset and progression of many human neurodegenerative diseases, meaning that a better understanding of the control of non-selective and selective autophagy pathways (including mitophagy) in disease-affected classes of neurons is needed. To achieve this, it is essential that the methodologies commonly used to study autophagy regulation under basal and stressed conditions in standard cell-line models are accurately applied when using hiPSC-derived neuronal cultures. Here, we discuss the roles and control of autophagy in human stem cells, and how autophagy contributes to neural differentiation in vitro. We also describe how autophagy-monitoring tools can be applied to hiPSC-derived neurons for the study of human neurodegenerative disease in vitro.

The Arl3 and Arl1 GTPases co-operate with Cog8 to regulate selective autophagy via Atg9 trafficking.

The Arl3-Arl1 GTPase cascade plays important roles in vesicle trafficking at the late Golgi and endosomes. Subunits of the conserved oligomeric Golgi (COG) complex, a tethering factor, are important for endosome-to-Golgi transport and contribute to the efficient functioning of the cytoplasm-to-vacuole targeting (Cvt) pathway, a well-known selective autophagy pathway. According to our findings, the Arl3-Arl1 GTPase cascade co-operates with Cog8 to regulate the Cvt pathway via Atg9 trafficking. arl3cog8Δ and arl1cog8Δ exhibit profound defects in aminopeptidase I maturation in rich medium. In addition, the Arl3-Arl1 cascade acts on the Cvt pathway via dynamic nucleotide binding. Furthermore, Atg9 accumulates at the late Golgi in arl3cog8Δ and arl1cog8Δ cells under normal growth conditions but not under starvation conditions. Thus, our results offer insight into the requirement for multiple components in the Golgi-endosome system to determine Atg9 trafficking at the Golgi, thereby regulating selective autophagy.

Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy.

The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

ABSTRACTSelective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

ATG8 Expansion: A Driver of Selective Autophagy Diversification?

Selective autophagy is a conserved homeostatic pathway that involves engulfment of specific cargo molecules into specialized organelles called autophagosomes. The ubiquitin-like protein ATG8 is a central player of the autophagy network that decorates autophagosomes and binds to numerous cargo receptors. Although highly conserved across eukaryotes, ATG8 diversified from a single protein in algae to multiple isoforms in higher plants. We present a phylogenetic overview of 376 ATG8 proteins across the green plant lineage that revealed family-specific ATG8 clades. Because these clades differ in fixed amino acid polymorphisms, they provide a mechanistic framework to test whether distinct ATG8 clades are functionally specialized. We propose that ATG8 expansion may have contributed to the diversification of selective autophagy pathways in plants.

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases.

Mechanistic insights into selective autophagy pathways: lessons from yeast.

Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery, the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation.

Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination.

Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker's yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker's yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker's yeast as a model to study the ubiquitin-dependent mitophagy pathway.

Parkin‐Mediated Mitophagy in Skeletal Muscle with Aging and Exercise

In skeletal muscle, the progressive age‐related loss of muscle mass and strength is known as sarcopenia. Alterations in skeletal muscle mitochondria are considered to be a contributing factor in this process. When mitochondria are no longer able to sustain the energetic requirements of the cell, selective autophagy (mitophagy) pathways are induced to promote the clearance of damaged mitochondria. In aged muscle, dysfunctional mitochondria generate increased amounts of reactive oxygen species (ROS) and can impair quality control mechanisms that facilitate their removal. It remains unclear if mitophagy mediates this reduced response in aged muscle. Parkin is an ubiquitin ligase involved in mitophagy, but little is known about its function with aging. To evaluate mitophagic signaling in aged muscle, young (3 months) and aged (18 months) Parkin deficient (KO) mice and wild‐type (WT) animals were assigned to three groups: control, acute exercise, or acute exercise plus 2 hours of recovery. Muscle mass was reduced by 46% and 65% respectively in aged Parkin KO and WT mice when compared to their young counterparts. Furthermore, aged WT animals displayed a 25% decrease in whole muscle cytochrome c oxidase activity. This was accompanied by a 40% reduction in State 3 (active) intermyofibrillar mitochondrial respiration in aged WT animals. In response to exercise, both aged Parkin KO and WT mice exhibited ~30% declines in running performance, and increased lactic acidosis by 2.1‐ and 1.4‐fold, respectively. Interestingly, basal mitophagy flux and subsarcolemmal mitochondrial localization of LC3II, p62 and ubiquitin were augmented in both young and aged Parkin KO muscle. Acute exercise increased mitochondrial flux in young animals, but reduced flux in older muscle. Our findings indicate that the expression of mitophagy proteins and their localization to mitochondria are not decreased in aged muscle, even in the absence of Parkin. However, attenuation of exercise‐induced mitophagy in response to aging may contribute to mitochondrial accumulation and dysfunction in aged muscle.Support or Funding InformationSupported by NSERC.

FLNC myofibrillar myopathy results from impaired autophagy and protein insufficiency.

Myofibrillar myopathy is a progressive muscle disease characterized by the disintegration of muscle fibers and formation of protein aggregates. Causative mutations have been identified in nine genes encoding Z-disk proteins, including the actin binding protein filamin C (FLNC). To investigate the mechanism of disease in FLNCW2710X myopathy we overexpressed fluorescently tagged FLNC or FLNCW2710X in zebrafish. Expression of FLNCW2710X causes formation of protein aggregates but surprisingly, our studies reveal that the mutant protein localizes correctly to the Z-disk and is capable of rescuing the fiber disintegration phenotype that results from FLNC knockdown. This demonstrates that the functions necessary for muscle integrity are not impaired, and suggests that it is the formation of protein aggregates and subsequent sequestration of FLNC away from the Z-disk that results in myofibrillar disintegration. Similar to those found in patients, the aggregates in FLNCW2710X expressing fish contain the co-chaperone BAG3. FLNC is a target of the BAG3-mediated chaperone assisted selective autophagy (CASA) pathway and therefore we investigated its role, and the role of autophagy in general, in clearing protein aggregates. We reveal that despite BAG3 recruitment to the aggregates they are not degraded via CASA. Additionally, recruitment of BAG3 is sufficient to block alternative autophagy pathways which would otherwise clear the aggregates. This blockage can be relieved by reducing BAG3 levels or by stimulating autophagy. This study therefore identifies both BAG3 reduction and autophagy promotion as potential therapies for FLNCW2710X myofibrillar myopathy, and identifies protein insufficiency due to sequestration, compounded by impaired autophagy, as the cause.

NBR1 helps autophagosomes take a bite out of focal adhesions

![Figure][1] Autophagosomes (black) target focal adhesions (magenta) at the leading edge of a migrating cell. [Kenific et al.][2] identify a selective autophagy pathway that promotes cell migration by enhancing the turnover of focal adhesions. Autophagy pathways target cytoplasmic

Parkin promotes proteasomal degradation of p62: implication of selective vulnerability of neuronal cells in the pathogenesis of Parkinson's disease.

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson’s disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0230-9) contains supplementary material, which is available to authorized users.

A selective autophagy pathway takes an unconventional route

Selective autophagy transports specific cytoplasmic materials into lysosomes/vacuoles. In the case of macroautophagy the selectivity is mediated by receptors, which usually link the cargos to the machinery that sequesters them into the forming autophagosome. In our recent work, we found that fission yeast Nbr1, a homolog of the mammalian macroautophagy receptor NBR1, acts together with an unconventional autophagy-associated cargo sequestration apparatus, the endosomal sorting complexes required for transport (ESCRTs), to deliver 2 hydrolytic enzymes from the cytosol to the vacuole lumen. In this pathway, which we term the Nbr1-mediated vacuolar targeting (NVT) pathway, soluble cargos transit through the multi-vesicular body (MVB), rather than the autophagosome, on their way to the vacuole. Our findings reveal a novel mode of action of macroautophagy receptors and broaden our understanding of ESCRT-mediated autophagy.

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

Ubiquitin-Dependent And Independent Signals In Selective Autophagy.

Selective autophagy regulates the abundance of specific cellular components via a specialized arsenal of factors, termed autophagy receptors, that target protein complexes, aggregates, and whole organelles into lysosomes. Autophagy receptors bind to LC3/GABARAP proteins on phagophore and autophagosome membranes, and recognize signals on cargoes to deliver them to autophagy. Ubiquitin (Ub), a well-known signal for the degradation of polypeptides in the proteasome, also plays an important role in the recognition of cargoes destined for selective autophagy. In addition, a variety of cargoes are committed to selective autophagy pathways by Ub-independent mechanisms employing protein-protein interaction motifs, Ub-like modifiers, and sugar- or lipid-based signals. In this article we summarize Ub-dependent and independent selective autophagy pathways, and discuss regulatory mechanisms and challenges for future studies.

Amino-terminal arginylation as a degradation signal for selective autophagy.

The ubiquitin-proteasome system and the autophagy lysosome system are the two major protein degradation machineries in eukaryotic cells. These two systems coordinate the removal of unwanted intracellular materials, but the mechanism by which they achieve this synchronization is largely unknown. The ubiquitination of substrates serves as a universal degradation signal for both systems. Our study revealed that the amino-terminal Arg, a canonical N-degron in the ubiquitin-proteasome system, also acts as a degradation signal in autophagy. We showed that many ER residents, such as BiP, contain evolutionally conserved arginylation permissive pro-N-degrons, and that certain inducers like dsDNA or proteasome inhibitors cause their translocation into the cytoplasm where they bind misfolded proteins and undergo amino-terminal arginylation by arginyl transferase 1 (ATE1). The amino-terminal Arg of BiP binds p62, which triggers p62 oligomerization and enhances p62-LC3 interaction, thereby stimulating autophagic delivery and degradation of misfolded proteins, promoting cell survival. This study reveals a novel ubiquitin-independent mechanism for the selective autophagy pathway, and provides an insight into how these two major protein degradation pathways communicate in cells to dispose the unwanted proteins. [BMB Reports 2015; 48(9): 487-488]

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway.

Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.

Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous in vivo protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.

PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.