scholarly journals Plasticity of Polyubiquitin Recognition as Lysosomal Targeting Signals by the Endosomal Sorting Machinery

2007 ◽  
Vol 18 (10) ◽  
pp. 3952-3965 ◽  
Author(s):  
Herve Barriere ◽  
Csilla Nemes ◽  
Kai Du ◽  
Gergely L. Lukacs
Keyword(s):  
Multivesicular Body ◽  
Invariant Chain ◽  
Endosomal Sorting ◽  
Cytoplasmic Tails ◽  
Lysosomal Sorting ◽  
Targeting Signals ◽  
Lysosomal Targeting ◽  
Necessary And Sufficient ◽  
Kinetics Of ◽  
Hepatocyte Growth

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.

scholarly journals AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

2012 ◽  
Vol 23 (18) ◽  
pp. 3612-3623 ◽  
Author(s):  
Michael R. Dores ◽  
May M. Paing ◽  
Huilan Lin ◽  
William A. Montagne ◽  
Adriano Marchese ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Early Endosomes ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

scholarly journals ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

2012 ◽  
Vol 197 (3) ◽  
pp. 407-419 ◽  
Author(s):  
Michael R. Dores ◽  
Buxin Chen ◽  
Huilan Lin ◽  
Unice J.K. Soh ◽  
May M. Paing ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interacting Protein ◽  
Kinase Substrate ◽  
Endosomal Sorting ◽  
Ubiquitin Binding ◽  
Lysosomal Sorting ◽  
Protease Activated Receptor 1 ◽  
Signaling Receptors ◽  
G Protein Coupled ◽  
Hepatocyte Growth

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)–0, –I, –II, and –III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor–regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III–dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4–ESCRT-III interacting protein, bound to a YPX3L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPXnL motifs.

scholarly journals Distinct Roles for Tsg101 and Hrs in Multivesicular Body Formation and Inward Vesiculation

2006 ◽  
Vol 17 (8) ◽  
pp. 3469-3483 ◽  
Author(s):  
M. Razi ◽  
C. E. Futter
Keyword(s):  
Growth Factor ◽  
Mammalian Cells ◽  
Susceptibility Gene ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Early Endosome ◽  
Endosomal Sorting ◽  
Epidermal Growth ◽  
Tumor Susceptibility Gene ◽  
Hepatocyte Growth

In mammalian cells, epidermal growth factor (EGF) stimulation promotes multivesicular body (MVB) formation and inward vesiculation within MVB. Annexin 1 is required for EGF-stimulated inward vesiculation but not MVB formation, demonstrating that MVB formation (the number of MVBs/unit cytoplasm) and inward vesiculation (the number of internal vesicles/MVB) are regulated by different mechanisms. Here, we show that EGF-stimulated MVB formation requires the tumor susceptibility gene, Tsg101, a component of the ESCRT (endosomal sorting complex required for transport) machinery. Depletion of Tsg101 potently inhibits EGF degradation and MVB formation and causes the vacuolar domains of the early endosome to tubulate. Although Tsg101 depletion inhibits MVB formation and alters the morphology of the early endosome in unstimulated cells, these effects are much greater after EGF stimulation. In contrast, depletion of hepatocyte growth factor receptor substrate (Hrs) only modestly inhibits EGF degradation, does not induce tubulation of the early endosome, and causes the generation of enlarged MVBs that retain the ability to fuse with the lysosome. Together, these results indicate that Tsg101 is required for the formation of stable vacuolar domains within the early endosome that develop into MVBs and Hrs is required for the accumulation of internal vesicles within MVBs and that both these processes are up-regulated by EGF stimulation.

scholarly journals His Domain Protein Tyrosine Phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

2021 ◽  
Author(s):  
Gabrielle Parkinson ◽  
Peristera Roboti ◽  
Ling Zhang ◽  
Sandra Taylor ◽  
Philip Woodman
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Multivesicular Body ◽  
Nucleotide Exchange ◽  
Protein Tyrosine ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Nucleotide Exchange Factor ◽  
Lysosomal Sorting ◽  
Domain Protein

His Domain Protein Tyrosine Phosphatase (HD-PTP) collaborates with Endosomal Sorting Complexes Required for Transport (ESCRTs) to sort endosomal cargo into intralumenal vesicles, forming the multivesicular body. Completion of multivesicular body sorting is accompanied by maturation of the endosome into a late endosome, an event that requires inactivation of the early endosomal GTPase, Rab5. Here we show that HD-PTP links ESCRT function with endosomal maturation. HD-PTP depletion prevents multivesicular body sorting, whilst also blocking cargo from exiting Rab5-rich endosomes. HD-PTP depleted cells contain hyperphosphorylated Rabaptin-5, a cofactor for the Rab5 guanine nucleotide exchange factor, Rabex-5, though HD-PTP is unlikely to directly dephosphorylate Rabaptin-5. In addition, HD-PTP depleted cells exhibit Rabaptin-5 dependent hyperactivation of Rab5. HD-PTP binds directly to Rabaptin-5, between its Rabex-5 and Rab5 binding domains. This binding reaction involves the ESCRT-0/ESCRT-III binding site in HD-PTP and is competed by an ESCRT-III peptide. Jointly, these findings indicate that HD-PTP may alternately scaffold ESCRTs and modulate Rabex-5/Rabaptin-5 activity, thereby helping to coordinate the completion of MVB sorting with endosomal maturation.

scholarly journals Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking

2021 ◽  
Author(s):  
Stefanie S. Schmieder ◽  
Raju Tatituri ◽  
Michael Anderson ◽  
Kate Kelly ◽  
Wayne I. Lencer
Keyword(s):  
Acyl Chain ◽  
Vesicular Trafficking ◽  
Structural Motif ◽  
Structural Basis ◽  
Membrane Organization ◽  
Endosomal Sorting ◽  
Lysosomal Sorting ◽  
Acyl Chains ◽  
Complex Sphingolipids ◽  
Endocytic Pathways

AbstractThe complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but how the cell discerns among the different ceramides to affect such sorting remains unknown. To address mechanism, we synthesized a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assessed their sorting among different endocytic pathways. We found that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictated lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14*-motif was cholesterol dependent. Perturbations of the C14*-motif by unsaturation enabled GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independently of cholesterol. Unsaturation occurring beyond the C14*-motif in very long acyl chains rescued lysosomal sorting. These results define a structural motif underlying membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.

scholarly journals Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

2004 ◽  
Vol 165 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Matthew N.J. Seaman
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Multivesicular Body ◽  
Carboxypeptidase Y ◽  
Reporter Protein ◽  
Rapid Degradation ◽  
Endosomal Sorting ◽  
Lysosome Biogenesis ◽  
Golgi Transport ◽  
Efficient Retrieval

fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called “retromer” that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei

2002 ◽  
Vol 115 (16) ◽  
pp. 3253-3263 ◽  
Author(s):  
David L. Alexander ◽  
Kevin J. Schwartz ◽  
Andrew E. Balber ◽  
James D. Bangs
Keyword(s):  
Cell Surface ◽  
Trypanosoma Brucei ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Developmentally Regulated ◽  
Lysosomal Membrane Protein ◽  
Targeting Signals ◽  
Lysosomal Targeting ◽  
Endocytic Activity ◽  
Regulated Trafficking

p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67ΔCD) or cytoplasmic/transmembrane domains (p67ΔTM) was monitored by pulse-chase,surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67ΔCD and p67ΔTM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

scholarly journals Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion

2018 ◽  
Vol 115 (19) ◽  
pp. E4396-E4405 ◽  
Author(s):  
Sebastian Bänfer ◽  
Dominik Schneider ◽  
Jenny Dewes ◽  
Maximilian T. Strauss ◽  
Sven-A. Freibert ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Direct Interaction ◽  
Multivesicular Body ◽  
Dominant Negative ◽  
Amino Terminus ◽  
Multivesicular Bodies ◽  
Dramatic Decrease ◽  
Endosomal Sorting ◽  
Galectin 3 ◽  
Binding Lectin

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

scholarly journals A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

2018 ◽  
Vol 115 (38) ◽  
pp. E8900-E8908 ◽  
Author(s):  
Jessica B. A. Sadler ◽  
Dawn M. Wenzel ◽  
Lauren K. Williams ◽  
Marta Guindo-Martínez ◽  
Steven L. Alam ◽  
...  
Keyword(s):  
Cancer Susceptibility ◽  
Genome Instability ◽  
Multivesicular Body ◽  
Mitotic Checkpoint ◽  
Body Protein ◽  
Endosomal Sorting ◽  
Daughter Cells ◽  
Dna Replication Stress ◽  
Human Polymorphism ◽  
Mitotic Stress

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the endosomal-sorting complexes required for transport (ESCRT) machinery and is tightly regulated by charged multivesicular body protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4C (rs35094336, CHMP4CT232) increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: The CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome missegregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.

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