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Harald Alfred Stenmark - One of the best experts on this subject based on the ideXlab platform.
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The Phosphatidylinositol 3-Phosphate-binding protein SNX4 controls ATG9A recycling and autophagy.
Journal of cell science, 2021Co-Authors: Anthony Ravussin, Sharon A. Tooze, Andreas Brech, Harald Alfred StenmarkAbstract:Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Although these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the Phosphatidylinositol 3-Phosphate (PI3P)-binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.This article has an associated First Person interview with the first author of the paper.
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The Phosphatidylinositol 3-Phosphate binding protein SNX4 controls ATG9A recycling and autophagy
2020Co-Authors: Anthony Ravussin, Sharon A. Tooze, Harald Alfred StenmarkAbstract:Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the Phosphatidylinositol 3-Phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the transmembrane autophagy machinery protein ATG9A from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. Altogether, we propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.
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Monitoring Phosphatidylinositol 3-Phosphate in Multivesicular Endosome Biogenesis
Methods in enzymology, 2014Co-Authors: Catherine Sem Wegner, Harald Alfred Stenmark, Kay Oliver Schink, Andreas BrechAbstract:The endocytic pathway comprises a variety of intracellular compartments that regulate sorting of internalized plasma membrane constituents as well as extracellular material. A major sorting station on this route is the early endosome, where internalized receptors destined for degradation are trafficked from the limiting membrane into the interior of the endosome by formation of intraluminal vesicles (ILVs). This invagination and budding process leads to the biogenesis of multivesicular endosomes (MVEs). The formation of ILVs depends on the sequential action of protein complexes that are partly recruited in a Phosphatidylinositol 3-Phosphate (PtdIns3P)-dependent manner. The underlying mechanisms of the biogenesis of MVEs are still not completely understood and it is therefore of great interest to study the sorting of PtdIns3P in this process. We are describing several methods to track these sorting events by both light and electron microscopy and combination of both methods.
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Phosphatidylinositol 3‐Phosphate, a lipid that regulates membrane dynamics, protein sorting and cell signalling
BioEssays : news and reviews in molecular cellular and developmental biology, 2013Co-Authors: Kay Oliver Schink, Camilla Raiborg, Harald Alfred StenmarkAbstract:Phosphatidylinositol 3-Phosphate (PtdIns3P) is generated on the cytosolic leaflet of cellular membranes, primarily by phosphorylation of Phosphatidylinositol by class II and class III Phosphatidylinositol 3-kinases. The bulk of this lipid is found on the limiting and intraluminal membranes of endosomes, but it can also be detected in domains of phagosomes, autophagosome precursors, cytokinetic bridges, the plasma membrane and the nucleus. PtdIns3P controls cellular functions through recruitment of specific protein effectors, many of which contain FYVE or PX domains. Cellular processes known to be controlled by PtdIns3P and its effectors include endosomal fusion, sorting and motility, autophagy, cytokinesis, regulated exocytosis and signal transduction. Here we discuss how Ptdins3P is generated on specific cellular membranes, how its localizations and functions can be studied, and how its effectors serve to control cellular functions. Editor's suggested further reading in BioEssays: Phosphatidylinositol 4,5-bisPhosphate: Targeted production and signaling Abstract How does SHIP1/2 balance PtdIns(3,4)P2 and does it signal independently of its phosphatase activity? Abstract Phosphatidylinositol-3,4,5-trisPhosphate: Tool of choice for class I PI 3-kinases Abstract Phosphatidylinositol-4-Phosphate: The Golgi and beyond Abstract
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Time-Resolved Ultrastructural Detection of Phosphatidylinositol 3-Phosphate:
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2010Co-Authors: Susanne Stuffers, Harald Alfred Stenmark, Kay Oliver Schink, Lene Malerød, Silvia Corvera, Andreas BrechAbstract:Phosphatidylinositol 3-Phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization. The probe localized to early and late endocytic compartments according to the time period of dimerization, which indicates that PtdIns(3)P is turned over via the endocytic machinery. In the functional context of epidermal growth factor (EGF) stimulation, we observed that dimerization of the probe led to clustering of mainly early endocytic structures, leaving most of the probe localized to the limiting membrane of endosomes. Interestingly, these clustered endosomes contained coats positive for the PtdIns(3)P-binding protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), indicating that the probe did not displace Hrs binding. We conclude that the dimerizer-inducible probe is useful for the time-resolved detection of PtdIns(3)P at the ultrastructural level, but its effects on endosome morphology after EGF stimulation need to be taken into account. (J Histochem Cytochem 58:1025–1032, 2010)
Margaret S Robinson - One of the best experts on this subject based on the ideXlab platform.
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rag gtpases and Phosphatidylinositol 3 Phosphate mediate recruitment of the ap 5 spg11 spg15 complex
Journal of Cell Biology, 2021Co-Authors: Jennifer Hirst, G Hesketh, Anneclaude Gingras, Margaret S RobinsonAbstract:Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.
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Rag GTPases and Phosphatidylinositol 3-Phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.
The Journal of cell biology, 2021Co-Authors: Jennifer Hirst, Anneclaude Gingras, Geoffrey G Hesketh, Margaret S RobinsonAbstract:Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.
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rag gtpases and Phosphatidylinositol 3 Phosphate mediate recruitment of the ap 5 spg11 spg15 complex
bioRxiv, 2020Co-Authors: Jennifer Hirst, G Hesketh, Anneclaude Gingras, Margaret S RobinsonAbstract:Abstract Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells, and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which on its own localises to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15 while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway, and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation. Summary The AP-5/SPG11/SPG15 complex is recruited onto late endosomes/lysosomes, and contributes to lysosomal homeostasis and autophagic lysosome reformation. Hirst et al. show that recruitment is by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate and Rag GTPases, thus uncovering a link between AP-5/SPG11/SPG15 and the mTORC1 pathway.
Janni Petersen - One of the best experts on this subject based on the ideXlab platform.
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Ste12/Fab1 Phosphatidylinositol-3-Phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control.
PloS one, 2017Co-Authors: David Cobley, Mirita Franz-wachtel, Karsten Krug, Boris Macek, Lenka Hálová, Marie Schauries, Adrian Kaczmarek, Janni PetersenAbstract:Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-Phosphatidylinositol-3-Phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division.
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ste12 fab1 Phosphatidylinositol 3 Phosphate 5 kinase is required for nitrogen regulated mitotic commitment and cell size control
PLOS ONE, 2017Co-Authors: David Cobley, Karsten Krug, Boris Macek, Janni Petersen, Lenka Hálová, Marie Schauries, Adrian Kaczmarek, Mirita FranzwachtelAbstract:Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-Phosphatidylinositol-3-Phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division.
David J. Gillooly - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylinositol 3-Phosphate is found in microdomains of early endosomes
Histochemistry and Cell Biology, 2003Co-Authors: David J. Gillooly, Camilla Raiborg, Harald Alfred StenmarkAbstract:Phosphatidylinositol 3-Phosphate [PI(3)P] is a Phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.
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Intracellular trafficking and turnover of Phosphatidylinositol 3-Phosphate
Seminars in Cell & Developmental Biology, 2001Co-Authors: Harald Alfred Stenmark, David J. GilloolyAbstract:Abstract Phosphatidylinositol 3-kinases (PI 3-kinases) regulate cellular functions through the 3′ -phosphorylation of Phosphatidylinositol (PI) and its derivatives. The PI 3-kinase product Phosphatidylinositol 3-Phosphate [PI(3)P] functions to recruit and activate effector proteins containing FYVE zinc finger domains. These proteins have various functions in endocytic membrane trafficking, cytoskeletal regulation and signal transduction. In order to understand the function of FYVE proteins, it is essential to study the formation, localisation, trafficking and turnover of PI(3)P. Here we review recent evidence that PI(3)P is formed on early endosomes through the activity of a PI 3-kinase which is recruited by the GTPase Rab5, and that the PI(3)P is subsequently internalised into intralumenal vesicles of multivesicular endosomes for turnover.
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localization of Phosphatidylinositol 3 Phosphate in yeast and mammalian cells
The EMBO Journal, 2000Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred StenmarkAbstract:Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, Phosphatidylinositol 3-Phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.
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localization of Phosphatidylinositol 3 Phosphate in yeast and mammalian cells
The EMBO Journal, 2000Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred StenmarkAbstract:Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, Phosphatidylinositol 3-Phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Δ yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.
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Localization of Phosphatidylinositol 3-Phosphate in yeast and mammalian cells.
The EMBO journal, 2000Co-Authors: David J. Gillooly, Jean Michel Gaullier, R J Gould, Nia J Bryant, Margaret R. Lindsay, Robert G. Parton, Isabel C Morrow, Harald Alfred StenmarkAbstract:Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, Phosphatidylinositol 3-Phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.
Anneclaude Gingras - One of the best experts on this subject based on the ideXlab platform.
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rag gtpases and Phosphatidylinositol 3 Phosphate mediate recruitment of the ap 5 spg11 spg15 complex
Journal of Cell Biology, 2021Co-Authors: Jennifer Hirst, G Hesketh, Anneclaude Gingras, Margaret S RobinsonAbstract:Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.
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Rag GTPases and Phosphatidylinositol 3-Phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.
The Journal of cell biology, 2021Co-Authors: Jennifer Hirst, Anneclaude Gingras, Geoffrey G Hesketh, Margaret S RobinsonAbstract:Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.
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rag gtpases and Phosphatidylinositol 3 Phosphate mediate recruitment of the ap 5 spg11 spg15 complex
bioRxiv, 2020Co-Authors: Jennifer Hirst, G Hesketh, Anneclaude Gingras, Margaret S RobinsonAbstract:Abstract Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells, and occurs by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which on its own localises to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15 while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway, and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation. Summary The AP-5/SPG11/SPG15 complex is recruited onto late endosomes/lysosomes, and contributes to lysosomal homeostasis and autophagic lysosome reformation. Hirst et al. show that recruitment is by coincidence detection, requiring both Phosphatidylinositol 3-Phosphate and Rag GTPases, thus uncovering a link between AP-5/SPG11/SPG15 and the mTORC1 pathway.