Journal articles: 'Endosomal Sorting Complexes Required for Transport'

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Nickerson, Daniel P., Matthew West, and Greg Odorizzi. "Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes." Journal of Cell Biology 175, no. 5 (November 27, 2006): 715–20. http://dx.doi.org/10.1083/jcb.200606113.

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The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.

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Bache, Kristi G., Andreas Brech, Anja Mehlum, and Harald Stenmark. "Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes." Journal of Cell Biology 162, no. 3 (August 4, 2003): 435–42. http://dx.doi.org/10.1083/jcb.200302131.

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Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.

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Dukes, Joseph D., Judith D. Richardson, Ruth Simmons, and Paul Whitley. "A dominant-negative ESCRT-III protein perturbs cytokinesis and trafficking to lysosomes." Biochemical Journal 411, no. 2 (March 27, 2008): 233–39. http://dx.doi.org/10.1042/bj20071296.

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In eukaryotic cells, the completion of cytokinesis is dependent on membrane trafficking events to deliver membrane to the site of abscission. Golgi and recycling endosomal-derived proteins are required for the terminal stages of cytokinesis. Recently, protein subunits of the ESCRT (endosomal sorting complexes required for transport) that are normally involved in late endosome to lysosome trafficking have also been implicated in abscission. Here, we report that a subunit, CHMP3 (charged multivesicular body protein-3), of ESCRT-III localizes at the midbody. Deletion of the C-terminal autoinhibitory domain of CHMP3 inhibits cytokinesis. At the midbody, CHMP3 does not co-localize with Rab11, suggesting that it is not present on recycling endosomes. These results combined provide compelling evidence that proteins involved in late endosomal function are necessary for the end stages of cytokinesis.

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Bache, Kristi G., Susanne Stuffers, Lene Malerød, Thomas Slagsvold, Camilla Raiborg, Delphine Lechardeur, Sébastien Wälchli, Gergely L. Lukacs, Andreas Brech, and Harald Stenmark. "The ESCRT-III Subunit hVps24 Is Required for Degradation but Not Silencing of the Epidermal Growth Factor Receptor." Molecular Biology of the Cell 17, no. 6 (June 2006): 2513–23. http://dx.doi.org/10.1091/mbc.e05-10-0915.

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The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

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Shideler, Tess, Daniel P. Nickerson, Alexey J. Merz, and Greg Odorizzi. "Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9." Molecular Biology of the Cell 26, no. 7 (April 2015): 1345–56. http://dx.doi.org/10.1091/mbc.e14-06-1156.

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Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization.

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Luzio, J. Paul, Michael D. J. Parkinson, Sally R. Gray, and Nicholas A. Bright. "The delivery of endocytosed cargo to lysosomes." Biochemical Society Transactions 37, no. 5 (September 21, 2009): 1019–21. http://dx.doi.org/10.1042/bst0371019.

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In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.

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Herz, Hans-Martin, and Andreas Bergmann. "Genetic analysis of ESCRT function in Drosophila: a tumour model for human Tsg101." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 204–7. http://dx.doi.org/10.1042/bst0370204.

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Class E Vps (vacuolar protein sorting) proteins are components of the ESCRTs (endosomal sorting complexes required for transport) which are required for protein sorting at the early endosome. Most of these genes have been identified and genetically characterized in yeast. Recent genetic studies in Drosophila have revealed the phenotypic consequences of loss of vps function in multicellular organisms. In the present paper, we review these studies and discuss a mechanism which may explain how loss of the human Tsg101 (tumour susceptibility gene 101), a vps23 orthologue, causes tumours.

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Davies, Brian A., Ishara F. Azmi, and David J. Katzmann. "Regulation of Vps4 ATPase activity by ESCRT-III." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 143–45. http://dx.doi.org/10.1042/bst0370143.

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MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

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Rodahl, Lina M., Susanne Stuffers, Viola H. Lobert, and Harald Stenmark. "The role of ESCRT proteins in attenuation of cell signalling." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 137–42. http://dx.doi.org/10.1042/bst0370137.

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The ESCRT (endosomal sorting complex required for transport) machinery consists of four protein complexes that mediate sorting of ubiquitinated membrane proteins into the intraluminal vesicles of multivesicular endosomes, thereby targeting them for degradation in lysosomes. In the present paper, we review how ESCRT-mediated receptor down-regulation affects signalling downstream of Notch and growth factor receptors, and how ESCRTs may control cell proliferation, survival and cytoskeletal functions and contribute to tumour suppression.

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Zhang, Junbing, Jinchao Liu, Anne Norris, Barth D. Grant, and Xiaochen Wang. "A novel requirement for ubiquitin-conjugating enzyme UBC-13 in retrograde recycling of MIG-14/Wntless and Wnt signaling." Molecular Biology of the Cell 29, no. 17 (August 15, 2018): 2098–112. http://dx.doi.org/10.1091/mbc.e17-11-0639.

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After endocytosis, transmembrane cargoes such as signaling receptors, channels, and transporters enter endosomes where they are sorted to different destinations. Retromer and ESCRT (endosomal sorting complex required for transport) are functionally distinct protein complexes on endosomes that direct cargo sorting into the recycling retrograde transport pathway and the degradative multivesicular endosome pathway (MVE), respectively. Cargoes destined for degradation in lysosomes are decorated with K63-linked ubiquitin chains, which serve as an efficient sorting signal for entry into the MVE pathway. Defects in K63-linked ubiquitination disrupt MVE sorting and degradation of membrane proteins. Here, we unexpectedly found that UBC-13, the E2 ubiquitin-conjugating enzyme that generates K63-linked ubiquitin chains, is essential for retrograde transport of multiple retromer-dependent cargoes including MIG-14/Wntless. Loss of ubc-13 disrupts MIG-14/Wntless trafficking from endosomes to the Golgi, causing missorting of MIG-14 to lysosomes and impairment of Wnt-dependent processes. We observed that retromer-associated SNX-1 and the ESCRT-0 subunit HGRS-1/Hrs localized to distinct regions on a common endosome in wild type but overlapped on ubc-13(lf) endosomes, indicating that UBC-13 is important for the separation of retromer and ESCRT microdomains on endosomes. Our data suggest that cargo ubiquitination mediated by UBC-13 plays an important role in maintaining the functionally distinct subdomains to ensure efficient cargo segregation on endosomes.

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Parkinson, Michael D. J., Siân C. Piper, Nicholas A. Bright, Jennifer L. Evans, Jessica M. Boname, Katherine Bowers, Paul J. Lehner, and J. Paul Luzio. "A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I." Biochemical Journal 471, no. 1 (September 21, 2015): 79–88. http://dx.doi.org/10.1042/bj20150336.

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Using RNAi, a non-canonical pathway of endosomal sorting complexes required for transport was identified that is responsible for sorting virally ubiquitinated MHC class I into multivesicular bodies (MVBs) during down-regulation of this protein from the cell surface.

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Schluter, Cayetana, Karen K. Y. Lam, Jochen Brumm, Bella W. Wu, Matthew Saunders, Tom H. Stevens, Jennifer Bryan, and Elizabeth Conibear. "Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex." Molecular Biology of the Cell 19, no. 4 (April 2008): 1282–94. http://dx.doi.org/10.1091/mbc.e07-07-0659.

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Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.

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Luzio, J. Paul, Sally R. Gray, and Nicholas A. Bright. "Endosome–lysosome fusion." Biochemical Society Transactions 38, no. 6 (November 24, 2010): 1413–16. http://dx.doi.org/10.1042/bst0381413.

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The delivery of endocytosed cargo to lysosomes occurs through kissing and direct fusion of late endosomes/MVBs (multivesicular bodies) and lysosomes. Live-cell and electron microscopy experiments together with cell-free assays have allowed us to describe the characteristics of the delivery process and determine the core protein machinery required for fusion. The ESCRT (endosomal sorting complex required for transport) machinery is required for MVB biogenesis. The HOPS (homotypic fusion and vacuole protein sorting) complex is required for endosome–lysosome tethering and a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex including the R-SNARE VAMP7 (vesicle-associated membrane protein 7) mediates endosome–lysosome membrane fusion. Protein-binding partners of VAMP7 including the clathrin adaptors AP-3 (adaptor protein 3) and Hrb (HIV Rev-binding protein) are required for its correct intracellular localization and function. Overall, co-ordination of the activities of ESCRT, HOPS and SNARE complexes are required for efficient delivery of endocytosed macromolecules to lysosomes. Endosome–lysosome fusion results in a hybrid organelle from which lysosomes are re-formed. Defects in fusion and/or lysosome reformation occur in a number of lysosome storage diseases.

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Morvan, Joëlle, Bruno Rinaldi, and Sylvie Friant. "Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes." Molecular Biology of the Cell 23, no. 20 (October 15, 2012): 4054–64. http://dx.doi.org/10.1091/mbc.e12-01-0001.

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Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27S613A, MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.

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Mattissek, Claudia, and David Teis. "The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis." Molecular Membrane Biology 31, no. 4 (March 18, 2014): 111–19. http://dx.doi.org/10.3109/09687688.2014.894210.

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Nickerson, Daniel P., Matthew West, Ryan Henry, and Greg Odorizzi. "Regulators of Vps4 ATPase Activity at Endosomes Differentially Influence the Size and Rate of Formation of Intralumenal Vesicles." Molecular Biology of the Cell 21, no. 6 (March 15, 2010): 1023–32. http://dx.doi.org/10.1091/mbc.e09-09-0776.

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Recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomes regulates selective inclusion of transmembrane proteins into the lumenal vesicles of multivesicular bodies (MVBs). ESCRT-0, -I, and -II bind directly to ubiquitinated transmembrane cargoes of the MVB pathway, whereas polymerization of ESCRT-III at endosomes is thought to bend the membrane and/or provide the energetic force that drives membrane scission and detachment of vesicles into the endosome lumen. Disassembly of the ESCRT-III polymer and dissociation of its subunits from endosomes requires the Vps4 ATPase, the activity of which is controlled in vivo by regulatory proteins. We identify distinct spatiotemporal roles for Vps4-regulating proteins through examinations of subcellular localization and endosome morphology. Did2 plays a unique role in the regulation of MVB lumenal vesicle size, whereas Vtal and Vps60 promote efficient membrane scission and delivery of membrane to the endosome lumen. These morphological effects probably result from Vps4-mediated manipulations of ESCRT-III, because we show dissociation of ESCRT-0, -I, and -II from endosomes is not directly dependent on Vps4 activity.

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Tabernero, Lydia, and Philip Woodman. "Dissecting the role of His domain protein tyrosine phosphatase/PTPN23 and ESCRTs in sorting activated epidermal growth factor receptor to the multivesicular body." Biochemical Society Transactions 46, no. 5 (September 6, 2018): 1037–46. http://dx.doi.org/10.1042/bst20170443.

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Sorting of activated epidermal growth factor receptor (EGFR) into intraluminal vesicles (ILVs) within the multivesicular body (MVB) is an essential step during the down-regulation of the receptor. The machinery that drives EGFR sorting attaches to the cytoplasmic face of the endosome and generates vesicles that bud into the endosome lumen, but somehow escapes encapsulation itself. This machinery is termed the ESCRT (endosomal sorting complexes required for transport) pathway, a series of multi-protein complexes and accessory factors first identified in yeast. Here, we review the yeast ESCRT pathway and describe the corresponding components in mammalian cells that sort EGFR. One of these is His domain protein tyrosine phosphatase (HD-PTP/PTPN23), and we review the interactions involving HD-PTP and ESCRTs. Finally, we describe a working model for how this ESCRT pathway might overcome the intrinsic topographical problem of EGFR sorting to the MVB lumen.

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Chu, Tony, Ji Sun, Suraj Saksena, and Scott D. Emr. "New component of ESCRT-I regulates endosomal sorting complex assembly." Journal of Cell Biology 175, no. 5 (December 4, 2006): 815–23. http://dx.doi.org/10.1083/jcb.200608053.

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The endosomal sorting complex required for transport (ESCRT) complexes play a critical role in receptor down-regulation and retroviral budding. Although the crystal structures of two ESCRT complexes have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery are still poorly understood. We identify a new component of the ESCRT-I complex, multivesicular body sorting factor of 12 kD (Mvb12), and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit vacuolar protein sorting 23 (Vps23). We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled-coil domain of Vps23, as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and -II in the cytosol. We propose that Mvb12 stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT-I and -II is spatially and temporally restricted to the surface of the endosome after activation of the MVB sorting reaction.

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Mageswaran, Shrawan Kumar, Natalie K. Johnson, Greg Odorizzi, and Markus Babst. "Constitutively active ESCRT-II suppresses the MVB-sorting phenotype of ESCRT-0 and ESCRT-I mutants." Molecular Biology of the Cell 26, no. 3 (February 2015): 554–68. http://dx.doi.org/10.1091/mbc.e14-10-1469.

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The endosomal sorting complex required for transport (ESCRT) protein complexes function at the endosome in the formation of intraluminal vesicles (ILVs) containing cargo proteins destined for the vacuolar/lysosomal lumen. The early ESCRTs (ESCRT-0 and -I) are likely involved in cargo sorting, whereas ESCRT-III and Vps4 function to sever the neck of the forming ILVs. ESCRT-II links these functions by initiating ESCRT-III formation in an ESCRT-I–regulated manner. We identify a constitutively active mutant of ESCRT-II that partially suppresses the phenotype of an ESCRT-I or ESCRT-0 deletion strain, suggesting that these early ESCRTs are not essential and have redundant functions. However, the ESCRT-III/Vps4 system alone is not sufficient for ILV formation but requires cargo sorting mediated by one of the early ESCRTs.

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Skalicky, Jack J., Jun Arii, Dawn M. Wenzel, William-May B. Stubblefield, Angela Katsuyama, Nathan T. Uter, Monika Bajorek, David G. Myszka, and Wesley I. Sundquist. "Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport." Journal of Biological Chemistry 287, no. 52 (October 26, 2012): 43910–26. http://dx.doi.org/10.1074/jbc.m112.417899.

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Hsiao, Jye-Chian, Li-Wei Chu, Yung-Tsun Lo, Sue-Ping Lee, Tzu-Jung Chen, Cheng-Yen Huang, Yueh-Hsin Ping, and Wen Chang. "Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22." Journal of Virology 89, no. 16 (June 3, 2015): 8365–82. http://dx.doi.org/10.1128/jvi.00209-15.

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ABSTRACTVaccinia virus, the prototype of theOrthopoxvirusgenus in the familyPoxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.IMPORTANCEVaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

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Gershlick, David C., Christina Schindler, Yu Chen, and Juan S. Bonifacino. "TSSC1 is novel component of the endosomal retrieval machinery." Molecular Biology of the Cell 27, no. 18 (September 15, 2016): 2867–78. http://dx.doi.org/10.1091/mbc.e16-04-0209.

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Endosomes function as a hub for multiple protein-sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. Two tethering complexes named GARP (composed of ANG2, VPS52, VPS53, and VPS54 subunits) and EARP (composed of ANG2, VPS52, VPS53, and Syndetin subunits) were previously shown to participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. Little is known, however, about other proteins that function with GARP and EARP in these processes. Here we identify a protein named TSSC1 as a specific interactor of both GARP and EARP and as a novel component of the endosomal retrieval machinery. TSSC1 is a predicted WD40/β-propeller protein that coisolates with both GARP and EARP in affinity purification, immunoprecipitation, and gel filtration analyses. Confocal fluorescence microscopy shows colocalization of TSSC1 with both GARP and EARP. Silencing of TSSC1 impairs transport of internalized Shiga toxin B subunit to the TGN, as well as recycling of internalized transferrin to the plasma membrane. Fluorescence recovery after photobleaching shows that TSSC1 is required for efficient recruitment of GARP to the TGN. These studies thus demonstrate that TSSC1 plays a critical role in endosomal retrieval pathways as a regulator of both GARP and EARP function.

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Urbé, S., J. McCullough, P. Row, I. A. Prior, R. Welchman, and M. J. Clague. "Control of growth factor receptor dynamics by reversible ubiquitination." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 754–56. http://dx.doi.org/10.1042/bst0340754.

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Activated tyrosine kinase receptors acquire ubiquitin tags. Ubiquitination governs receptor down-regulation through interaction with components of the endosomal ESCRT (endosomal sorting complexes required for transport) machinery that shepherds receptors into luminal vesicles of multivesicular bodies en route to the lysosome. We have characterized two de-ubiquitinating enzymes that interact with components of this machinery. AMSH [associated molecule with the SH3 domain (Src homology 3 domain) of STAM (signal transducing adapter molecule)] shows specificity for Lys63- over Lys48-linked ubiquitin and may act to rescue receptors from taking the lysosomal pathway. In contrast, UBPY (ubiquitin-specific processing protease Y) does not discriminate between Lys48 and Lys63-linked chains and is required for lysosomal sorting.

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Rue, Sarah M., Sara Mattei, Suraj Saksena, and Scott D. Emr. "Novel Ist1-Did2 Complex Functions at a Late Step in Multivesicular Body Sorting." Molecular Biology of the Cell 19, no. 2 (February 2008): 475–84. http://dx.doi.org/10.1091/mbc.e07-07-0694.

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In Saccharomyces cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport. Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo-sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Δ is synthetic with vta1Δ and vps60Δ, indicating that Ist1 is also a positive component of the MVB-sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Δ mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB-sorting pathway.

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Woodman, Philip. "ESCRT-III on endosomes: new functions, new activation pathway." Biochemical Journal 473, no. 2 (January 5, 2016): e5-e8. http://dx.doi.org/10.1042/bj20151115.

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The multivesicular body (MVB) pathway sorts ubiquitinated membrane cargo to intraluminal vesicles (ILVs) within the endosome, en route to the lysosomal lumen. The pathway involves the sequential action of conserved protein complexes [endosomal sorting complexes required for transport (ESCRTs)], culminating in the activation by ESCRT-II of ESCRT-III, a membrane-sculpting complex. Although this linear pathway of ESCRT activation is widely accepted, a study by Luzio and colleagues in a recent issue of the Biochemical Journal suggests that there is greater complexity in ESCRT-III activation, at least for some MVB cargoes. They show that ubiquitin-dependent sorting of major histocompatibility complex (MHC) class I to the MVB requires the central ESCRT-III complex but does not involve either ESCRT-II or functional links between ESCRT-II and ESCRT-III. Instead, they propose that MHC class I utilizes histidine-domain protein tyrosine phosphatase (HD-PTP), a non-canonical ESCRT interactor, to promote ESCRT-III activation.

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Takemoto, Kodai, Kazuo Ebine, Jana Christin Askani, Falco Krüger, Zaida Andrés Gonzalez, Emi Ito, Tatsuaki Goh, Karin Schumacher, Akihiko Nakano, and Takashi Ueda. "Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis." Proceedings of the National Academy of Sciences 115, no. 10 (February 20, 2018): E2457—E2466. http://dx.doi.org/10.1073/pnas.1717839115.

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Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.

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Shields, S. Brookhart, Andrea J. Oestreich, Stanley Winistorfer, Doris Nguyen, Johanna A. Payne, David J. Katzmann, and Robert Piper. "ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting." Journal of Cell Biology 185, no. 2 (April 20, 2009): 213–24. http://dx.doi.org/10.1083/jcb.200811130.

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Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.

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Neto, Hélia, Alexandra Kaupisch, Louise L. Collins, and Gwyn W. Gould. "Syntaxin 16 is a master recruitment factor for cytokinesis." Molecular Biology of the Cell 24, no. 23 (December 2013): 3663–74. http://dx.doi.org/10.1091/mbc.e13-06-0302.

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Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome–associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

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Tu, C., C. F. Ortega-Cava, P. Winograd, M. J. Stanton, A. L. Reddi, I. Dodge, R. Arya, et al. "Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions." Proceedings of the National Academy of Sciences 107, no. 37 (August 30, 2010): 16107–12. http://dx.doi.org/10.1073/pnas.1009471107.

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Gingras, Marie-Claude, Jalal M. Kazan, and Arnim Pause. "Role of ESCRT component HD-PTP/PTPN23 in cancer." Biochemical Society Transactions 45, no. 3 (June 15, 2017): 845–54. http://dx.doi.org/10.1042/bst20160332.

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Sustained cellular signalling originated from the receptors located at the plasma membrane is widely associated with cancer susceptibility. Endosomal sorting and degradation of the cell surface receptors is therefore crucial to preventing chronic downstream signalling and tumorigenesis. Since the Endosomal Sorting Complexes Required for Transport (ESCRT) controls these processes, ESCRT components were proposed to act as tumour suppressor genes. However, the bona fide role of ESCRT components in tumorigenesis has not been clearly demonstrated. The ESCRT member HD-PTP/PTPN23 was recently identified as a novel haplo-insufficient tumour suppressor in vitro and in vivo, in mice and humans. In this mini-review, we outline the role of the ESCRT components in cancer and summarize the functions of HD-PTP/PTPN23 in tumorigenesis.

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Hurley, James H., and Xuefeng Ren. "The circuitry of cargo flux in the ESCRT pathway." Journal of Cell Biology 185, no. 2 (April 20, 2009): 185–87. http://dx.doi.org/10.1083/jcb.200903013.

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The endosomal sorting complex required for transport (ESCRT) complexes sort ubiquitinated membrane proteins into multivesicular bodies, which is a key step in the lysosomal degradation pathway. Shields et al. (Shields, S.B., A.J. Oestreich, S. Winistorfer, D. Nguyen, J.A. Payne, D.J. Katzmann, and R. Piper. 2009. J. Cell Biol. 185:213–224) identify a new ubiquitin-binding site in ESCRT-I and provide evidence that the upstream ESCRT-I and -II complexes sort cargo in parallel rather than in series.

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Xie, Qiurong, Ahai Chen, Wenhui Zheng, Huaijian Xu, Wenjie Shang, Huawei Zheng, Dongmei Zhang, et al. "Endosomal sorting complexes required for transport-0 is essential for fungal development and pathogenicity inFusarium graminearum." Environmental Microbiology 18, no. 11 (June 2, 2016): 3742–57. http://dx.doi.org/10.1111/1462-2920.13296.

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Boura, Evzen, Vassili Ivanov, Lars-Anders Carlson, Kiyoshi Mizuuchi, and James H. Hurley. "Endosomal Sorting Complex Required for Transport (ESCRT) Complexes Induce Phase-separated Microdomains in Supported Lipid Bilayers." Journal of Biological Chemistry 287, no. 33 (June 20, 2012): 28144–51. http://dx.doi.org/10.1074/jbc.m112.378646.

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Lee, Il-Hyung, Hiroyuki Kai, Lars-Anders Carlson, Jay T. Groves, and James H. Hurley. "Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15892–97. http://dx.doi.org/10.1073/pnas.1518765113.

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The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.

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Woodman, Philip. "ESCRT proteins, endosome organization and mitogenic receptor down-regulation." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 146–50. http://dx.doi.org/10.1042/bst0370146.

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Mitogenic tyrosine kinase receptors such as the EGFR (epidermal growth factor receptor) are endocytosed once they are activated at the cell surface. After reaching the early endosome, they are ubiquitinated within their cytosolic domain and are consequently sorted away from recycling receptors. They are then incorporated into intraluminal vesicles within the MVB (multivesicular body) en route to the lysosome, where they are degraded. MVB formation requires the stabilization of the vacuolar domain of the early endosome, the segregation of degradative cargo within this domain (with subsequent incorporation of receptors such as EGFR into intraluminal vesicles) and the physical separation and movement of this domain away from the tubular regions of the early endosome. How these different aspects of MVB biogenesis are coupled is unknown, but ESCRTs (endosomal sorting complexes required for transport) have been identified as key molecular players in driving mitogenic receptor sequestration and formation of intraluminal vesicles. The present review summarizes recent findings within the field and from our laboratory regarding the detailed function of ESCRTs and associated proteins in driving the ubiquitin-dependent sorting of EGFR and in maintaining the domain organization of the early endosome.

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Bache, Kristi G., Thomas Slagsvold, Alicia Cabezas, Ken R. Rosendal, Camilla Raiborg, and Harald Stenmark. "The Growth-Regulatory Protein HCRP1/hVps37A Is a Subunit of Mammalian ESCRT-I and Mediates Receptor Down-Regulation." Molecular Biology of the Cell 15, no. 9 (September 2004): 4337–46. http://dx.doi.org/10.1091/mbc.e04-03-0250.

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The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III. ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37. Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified. Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs. The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins. HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes. Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28. Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation. Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors.

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Meng, Bo, and Andrew M. L. Lever. "The Interplay between ESCRT and Viral Factors in the Enveloped Virus Life Cycle." Viruses 13, no. 2 (February 20, 2021): 324. http://dx.doi.org/10.3390/v13020324.

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Viruses are obligate parasites that rely on host cellular factors to replicate and spread. The endosomal sorting complexes required for transport (ESCRT) system, which is classically associated with sorting and downgrading surface proteins, is one of the host machineries hijacked by viruses across diverse families. Knowledge gained from research into ESCRT and viruses has, in turn, greatly advanced our understanding of many other cellular functions in which the ESCRT pathway is involved, e.g., cytokinesis. This review highlights the interplay between the ESCRT pathway and the viral factors of enveloped viruses with a special emphasis on retroviruses.

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Sevrioukov, Evgueni A., Nabil Moghrabi, Mary Kuhn, and Helmut Krämer. "A Mutation in dVps28 Reveals a Link between a Subunit of the Endosomal Sorting Complex Required for Transport-I Complex and the Actin Cytoskeleton in Drosophila." Molecular Biology of the Cell 16, no. 5 (May 2005): 2301–12. http://dx.doi.org/10.1091/mbc.e04-11-1013.

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Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. Here, we characterize a mutation in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated.

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Wemmer, Megan, Ishara Azmi, Matthew West, Brian Davies, David Katzmann, and Greg Odorizzi. "Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast." Journal of Cell Biology 192, no. 2 (January 24, 2011): 295–306. http://dx.doi.org/10.1083/jcb.201007018.

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Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.

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López-Reyes, Israel, Guillermina García-Rivera, Cecilia Bañuelos, Silvia Herranz, Olivier Vincent, César López-Camarillo, Laurence A. Marchat, and Esther Orozco. "Detection of the Endosomal Sorting Complex Required for Transport inEntamoeba histolyticaand Characterization of the EhVps4 Protein." Journal of Biomedicine and Biotechnology 2010 (2010): 1–15. http://dx.doi.org/10.1155/2010/890674.

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Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes inEntamoeba histolytica. We cloned and expressed theEhvps4gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 inE. histolyticaphagocytosis and virulence.

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Norris, Anne, Prasad Tammineni, Simon Wang, Julianne Gerdes, Alexandra Murr, Kelvin Y. Kwan, Qian Cai, and Barth D. Grant. "SNX-1 and RME-8 oppose the assembly of HGRS-1/ESCRT-0 degradative microdomains on endosomes." Proceedings of the National Academy of Sciences 114, no. 3 (January 4, 2017): E307—E316. http://dx.doi.org/10.1073/pnas.1612730114.

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After endocytosis, transmembrane cargo reaches endosomes, where it encounters complexes dedicated to opposing functions: recycling and degradation. Microdomains containing endosomal sorting complexes required for transport (ESCRT)-0 component Hrs [hepatocyte growth factor-regulated tyrosine kinase substrate (HGRS-1) in Caenorhabditis elegans] mediate cargo degradation, concentrating ubiquitinated cargo and organizing the activities of ESCRT. At the same time, retromer associated sorting nexin one (SNX-1) and its binding partner, J-domain protein RME-8, sort cargo away from degradation, promoting cargo recycling to the Golgi. Thus, we hypothesized that there could be important regulatory interactions between retromer and ESCRT that balance degradative and recycling functions. Taking advantage of the naturally large endosomes of the C. elegans coelomocyte, we visualized complementary ESCRT-0 and RME-8/SNX-1 microdomains in vivo and assayed the ability of retromer and ESCRT microdomains to regulate one another. We found in snx-1(0) and rme-8(ts) mutants increased endosomal coverage and intensity of HGRS-1–labeled microdomains, as well as increased total levels of HGRS-1 bound to membranes. These effects are specific to SNX-1 and RME-8, as loss of other retromer components SNX-3 and vacuolar protein sorting-associated protein 35 (VPS-35) did not affect HGRS-1 microdomains. Additionally, knockdown of hgrs-1 had little to no effect on SNX-1 and RME-8 microdomains, suggesting directionality to the interaction. Separation of the functionally distinct ESCRT-0 and SNX-1/RME-8 microdomains was also compromised in the absence of RME-8 and SNX-1, a phenomenon we observed to be conserved, as depletion of Snx1 and Snx2 in HeLa cells also led to greater overlap of Rme-8 and Hrs on endosomes.

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Rubio-Texeira, Marta, and Chris A. Kaiser. "Amino Acids Regulate Retrieval of the Yeast General Amino Acid Permease from the Vacuolar Targeting Pathway." Molecular Biology of the Cell 17, no. 7 (July 2006): 3031–50. http://dx.doi.org/10.1091/mbc.e05-07-0669.

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Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

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Bae, Donghwi, Kristin A. Moore, Jessica M. Mella, Samantha Y. Hayashi, and Julie Hollien. "Degradation of Blos1 mRNA by IRE1 repositions lysosomes and protects cells from stress." Journal of Cell Biology 218, no. 4 (February 20, 2019): 1118–27. http://dx.doi.org/10.1083/jcb.201809027.

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Cells respond to stress in the ER by initiating the widely conserved unfolded protein response. Activation of the ER transmembrane nuclease IRE1 leads to the degradation of specific mRNAs, but how this pathway affects the ability of cells to recover from stress is not known. Here, we show that degradation of the mRNA encoding biogenesis of lysosome-related organelles 1 subunit 1 (Blos1) leads to the repositioning of late endosomes (LEs)/lysosomes to the microtubule-organizing center in response to stress in mouse cells. Overriding Blos1 degradation led to ER stress sensitivity and the accumulation of ubiquitinated protein aggregates, whose efficient degradation required their independent trafficking to the cell center and the LE-associated endosomal sorting complexes required for transport. We propose that Blos1 regulation by IRE1 promotes LE-mediated microautophagy of protein aggregates and protects cells from their cytotoxic effects.

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Metcalf, Daniel, and Adrian M. Isaacs. "The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes." Biochemical Society Transactions 38, no. 6 (November 24, 2010): 1469–73. http://dx.doi.org/10.1042/bst0381469.

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ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.

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Li, Wei-Wei, Ying Nie, Yan Yang, Yong Ran, Wei-Wei Luo, Mei-Guang Xiong, Su-Yun Wang, Zhi-Sheng Xu, and Yan-Yi Wang. "Ubiquitination of TLR3 by TRIM3 signals its ESCRT-mediated trafficking to the endolysosomes for innate antiviral response." Proceedings of the National Academy of Sciences 117, no. 38 (September 2, 2020): 23707–16. http://dx.doi.org/10.1073/pnas.2002472117.

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Trafficking of toll-like receptor 3 (TLR3) from the endoplasmic reticulum (ER) to endolysosomes and its subsequent proteolytic cleavage are required for it to sense viral double-stranded RNA (dsRNA) and trigger antiviral response, yet the underlying mechanisms remain enigmatic. We show that the E3 ubiquitin ligase TRIM3 is mainly located in the Golgi apparatus and transported to the early endosomes upon stimulation with the dsRNA analog poly(I:C). TRIM3 mediates K63-linked polyubiquitination of TLR3 at K831, which is enhanced following poly(I:C) stimulation. The polyubiquitinated TLR3 is recognized and sorted by the ESCRT (endosomal sorting complex required for transport) complexes to endolysosomes. Deficiency of TRIM3 impairs TLR3 trafficking from the Golgi apparatus to endosomes and its subsequent activation.Trim3−/−cells and mice express lower levels of antiviral genes and show lower levels of inflammatory response following poly(I:C) but not lipopolysaccharide (LPS) stimulation. These findings suggest that TRIM3-mediated polyubiquitination of TLR3 represents a feedback-positive regulatory mechanism for TLR3-mediated innate immune and inflammatory responses.

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Stringer, Daniel K., and Robert C. Piper. "A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination." Journal of Cell Biology 192, no. 2 (January 17, 2011): 229–42. http://dx.doi.org/10.1083/jcb.201008121.

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ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.

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Hurley, James H., Young Jun Im, Hyung Ho Lee, Xuefeng Ren, Thomas Wollert, and Dong Yang. "Piecing together the ESCRTs." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 161–66. http://dx.doi.org/10.1042/bst0370161.

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High-resolution structural analysis has characterized nearly all of the individual domains of ESCRT (endosomal sorting complex required for transport) subunits, all of the core structures of the soluble complexes and many of the interactions involving domains. Recent emphasis in structural studies has shifted towards efforts to integrate these structures into a larger-scale model. Molecular simulations, hydrodynamic analysis, small-angle X-ray scattering and cryo-EM (electron microscopy) techniques have all been brought to bear on the ESCRT system over the last year.

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Nayak, Ramesh C., Shiva Keshava, Usha Pendurthi, and L. Vijaya Mohan Rao. "Role of Rab Proteins In Endocytosis and Trafficking of Factor VIIa and Endothelial Cell Protein C Receptor In Endothelial Cells." Blood 116, no. 21 (November 19, 2010): 1145. http://dx.doi.org/10.1182/blood.v116.21.1145.1145.

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Abstract Abstract 1145 Recent studies from our laboratory and others showed that endothelial cell protein C receptor (EPCR), the cellular receptor for protein C and activated protein C (APC), also serves as a receptor for factor VII (FVII) and activated factor VII (FVIIa). At present, the physiological importance of FVII/FVIIa binding to EPCR is largely unknown, but this interaction may play a role in the clearance or transport of FVII/FVIIa from circulation to tissues. Our recent studies showed that FVIIa (or APC) binding to EPCR promoted the endocytosis of EPCR via dynamin and caveolar-dependent pathways, and the endocytosed receptor-ligand complexes were accumulated in the recycling compartment (REC) before being targeted back to the cell surface (Blood 2009;114:1974-1986). Rab GTPases (Rab 4, Rab 5, Rab 7 and Rab 11 etc.), which localize to specific endosomal structures, have been shown to play crucial roles in the endocytic and exocytic pathways of receptor or receptor/ligand complexes. The role of these Ras-like small GTPases is unknown in endocytosis and trafficking of EPCR and EPCR/FVIIa complexes. The present study was undertaken in order to investigate the role of different Rab GTPases (Rab 4A, Rab 5 and Rab11) in the intracellular trafficking of EPCR and internalized FVIIa in endothelial cells. For this, we examined the effect of expressing wild-type (wt) or mutant Rab proteins on the intracellular distribution of FVIIa in human umbilical vein endothelial cells (HUVEC). The wild-type, constitutively active and dominant negative mutants of Rab 4A, Rab 5 and Rab 11 were cloned in adenoviral shuttle vector pacAd5 K-N pA CMV and the recombinant adenoviruses expressing these Rab GTPase variants were generated in human embryonic kidney (HEK) cells. HUVEC were infected with recombinant adenoviruses encoding for the wild-type, active or dominant negative mutant of Rab 4A, Rab 5 and Rab 11 (25 moi/cell). After culturing the cells for 24 h, they were incubated with recombinant FVIIa conjugated with Alexa fluor 488 fluorescent dye (AF488-FVIIa) for 1 h at 37°C. The intracellular distribution of FVIIa was analyzed by monitoring the fluorescence of AF488-FVIIa by confocal microscopy. The intracellular distribution of EPCR and Rab proteins was evaluated by confocal microscopy after immunofluorescence staining. Expression of Rab 4A wt or constitutively active Rab 4A (Q67L) forms led to accumulation of AF488-FVIIa within the Rab 4A positive early/sorting endosomes, whereas FVIIa accumulation in the REC was inhibited. In cells expressing Rab 4A dominant negative form (S22N), FVIIa was trafficked normally and accumulated in the REC. Rab 4A is known to regulate fusion of early and sorting endosomes, as well as recycling of the internalized receptor or receptor/ligand complexes from early/sorting endosomes back to the cell surface. Increased accumulation of FVIIa in early/sorting endosomes but a decrease in REC in HUVEC transduced to express wt and constitutively active Rab 4A, suggests that Rab 4A plays a role in the transport of internalized FVIIa and FVIIa-EPCR complexes from sorting endosomes back to the cell surface. HUVEC expressing Rab 5 wt or active mutant (Q79L) showed larger endosomal structures beneath the plasma membrane where EPCR and FVIIa were accumulated; very little FVIIa entered the REC. The trafficking of internalized FVIIa remained unaffected in HUVEC expressing Rab 5A dominant negative form (S34N). As Rab 5 is known to induce receptor internalization and fusion between early endosomes, the large endosomal structures containing AF488-FVIIa found in HUVEC expressing wt or constitutively active form but not in cells expressing the dominant negative form suggests that Rab 5 facilitates internalization of FVIIa-EPCR complexes. In contrast to the data obtained in HUVEC expressing Rab 4A and Rab 5, the intracellular trafficking of AF488-FVIIa remained unaffected in HUVEC expressing either wt or constitutively active Rab 11 mutant. Rab 11 dominant negative mutant (S34N) prevented the entry of AF488-FVIIa into REC. The observation that the dominant negative form of Rab 11 inhibits the entry of internalized FVIIa to the REC indicates that the activation of Rab 11 by GTP is required for the transport of FVIIa from sorting endosomes toward the recycling compartment. Overall our present data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR and internalized FVIIa in endothelial cells. Disclosures: No relevant conflicts of interest to declare.

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Adell, Manuel Alonso Y., Georg F. Vogel, Mehrshad Pakdel, Martin Müller, Herbert Lindner, Michael W. Hess, and David Teis. "Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation." Journal of Cell Biology 205, no. 1 (April 7, 2014): 33–49. http://dx.doi.org/10.1083/jcb.201310114.

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Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and –II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA–adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.

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Ghazi-Tabatabai, Sara, Takayuki Obita, Ajaybabu V. Pobbati, Olga Perisic, Rachel Y. Samson, Stephen D. Bell, and Roger L. Williams. "Evolution and assembly of ESCRTs." Biochemical Society Transactions 37, no. 1 (January 20, 2009): 151–55. http://dx.doi.org/10.1042/bst0370151.

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The AAA (ATPase associated with various cellular activities) proteins participate in membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA Vps (vacuolar protein sorting) 4 is central to traffic to lysosomes, retroviral budding and mammalian cell division. It dissociates ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes, enabling their recycling to the cytosol, and plays a role in fission of intraluminal vesicles within MVBs (multivesicular bodies). The mechanism of Vps4-catalysed disassembly of ESCRT networks is unknown; however, it requires interaction between Vps4 and ESCRT-III subunits. The 30 C-terminal residues of Vps2 and Vps46 (Did2) subunits are both necessary and sufficient for interaction with the Vps4 N-terminal MIT (microtubule-interacting and transport) domain, and the crystal structure of the Vps2 C-terminus in a complex with the Vps4 MIT domain shows that MIT helices α2 and α3 recognize a (D/E)XXLXXRLXXL(K/R) MIM (MIT-interacting motif). These Vps2–MIT interactions are essential for vacuolar sorting and for Vps4-catalysed disassembly of ESCRT-III networks in vitro. Electron microscopy of ESCRT-III filaments assembled in vitro has enabled us to identify surfaces of the Vps24 subunit that are critical for protein sorting in vivo. The ESCRT-III–Vps4 interaction predates the divergence of Archaea and Eukarya. The Crenarchaea have three classes of ESCRT-III-like subunits, and one of these subunits interacts with an archaeal Vps4-like protein in a manner closely related to the human Vps4–human ESCRT-III subunit Vps20 interaction. This archaeal Vps4–ESCRT-III interaction appears to have a fundamental role in cell division in the Crenarchaea.

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Journal articles on the topic 'Endosomal Sorting Complexes Required for Transport'

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