scholarly journals Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Jennifer Hirst ◽  
Geoffrey G. Hesketh ◽  
Anne-Claude Gingras ◽  
Margaret S. Robinson
Keyword(s):  
Protein Complex ◽  
Adaptor Protein ◽  
Coincidence Detection ◽  
Rag Gtpases ◽  
Fyve Domain ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Mtorc1 Pathway ◽  
Phosphatidylinositol 3

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.

scholarly journals Rag GTPases and Phosphatidylinositol 3-Phosphate Mediate Recruitment of the AP-5/SPG11/SPG15 Complex

2020 ◽  
Author(s):  
Jennifer Hirst ◽  
Geoffrey G. Hesketh ◽  
Anne-Claude Gingras ◽  
Margaret S. Robinson
Keyword(s):  
Protein Complex ◽  
Adaptor Protein ◽  
Coincidence Detection ◽  
Rag Gtpases ◽  
Fyve Domain ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Mtorc1 Pathway ◽  
Phosphatidylinositol 3

AbstractAdaptor 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.SummaryThe 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.

scholarly journals Sorting to Synaptic-like Microvesicles from Early and Late Endosomes Requires Overlapping but Not Identical Targeting Signals

2000 ◽  
Vol 11 (5) ◽  
pp. 1801-1814 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Daniel F. Cutler
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Protein Complex ◽  
Adaptor Protein ◽  
Neuroendocrine Cells ◽  
Dependent Manner ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Epidermal Growth

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.

scholarly journals Numb regulates the balance between Notch recycling and late-endosome targeting inDrosophilaneural progenitor cells

2016 ◽  
Vol 27 (18) ◽  
pp. 2857-2866 ◽  
Author(s):  
Seth A. Johnson ◽  
Diana Zitserman ◽  
Fabrice Roegiers
Keyword(s):  
Notch Signaling ◽  
Asymmetric Cell Division ◽  
Adaptor Protein ◽  
Notch Signaling Pathway ◽  
Notch Receptor ◽  
Recycling Endosome ◽  
Notch Receptors ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Sensory Organ Precursor

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

scholarly journals Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

2015 ◽  
Vol 112 (15) ◽  
pp. 4636-4641 ◽  
Author(s):  
Andreas Jeschke ◽  
Nicole Zehethofer ◽  
Buko Lindner ◽  
Jessica Krupp ◽  
Dominik Schwudke ◽  
...  
Keyword(s):  
Early Endosome ◽  
Phagosome Maturation ◽  
Phagocytic Cells ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Lipid Kinases ◽  
Phosphatidylinositol 4 ◽  
Endocytic Compartments ◽  
Late Stages ◽  
Phosphatidylinositol 3

Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.

scholarly journals The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy

2021 ◽  
Vol 134 (3) ◽  
pp. jcs250670 ◽  
Author(s):  
Anthony Ravussin ◽  
Andreas Brech ◽  
Sharon A. Tooze ◽  
Harald Stenmark
Keyword(s):  
Membrane Protein ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Protein Traffic ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Phosphate Binding Protein ◽  
Golgi Network ◽  
Phosphatidylinositol 3

ABSTRACTLate 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.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

scholarly journals The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy

2020 ◽  
Author(s):  
Anthony Ravussin ◽  
Sharon A. Tooze ◽  
Harald Stenmark
Keyword(s):  
Membrane Protein ◽  
Autophagic Flux ◽  
Dependent Manner ◽  
Phosphate Binding ◽  
Protein Traffic ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Phosphate Binding Protein ◽  
Golgi Network ◽  
Phosphatidylinositol 3

AbstractLate 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.

scholarly journals BLOC-1 Is Required for Cargo-specific Sorting from Vacuolar Early Endosomes toward Lysosome-related Organelles

2007 ◽  
Vol 18 (3) ◽  
pp. 768-780 ◽  
Author(s):  
Subba Rao Gangi Setty ◽  
Danièle Tenza ◽  
Steven T. Truschel ◽  
Evelyn Chou ◽  
Elena V. Sviderskaya ◽  
...  
Keyword(s):  
Protein Complex ◽  
Genetic Disorder ◽  
Adaptor Protein ◽  
Transport Pathways ◽  
Cargo Transport ◽  
Early Endosomes ◽  
Hermansky Pudlak Syndrome ◽  
Lysosome Related Organelles ◽  
And Function ◽  
Tyrosinase Related Protein

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1–deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.

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