Charged Multivesicular Body Protein 2B (CHMP2B) of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) Polymerizes into Helical Structures Deforming the Plasma Membrane

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805504115 ◽

2018 ◽

Vol 115 (38) ◽

pp. E8900-E8908 ◽

Cited By ~ 19

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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Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

The Journal of Cell Biology ◽

10.1083/jcb.200707031 ◽

2008 ◽

Vol 180 (2) ◽

pp. 389-402 ◽

Cited By ~ 314

Author(s):

Phyllis I. Hanson ◽

Robyn Roth ◽

Yuan Lin ◽

John E. Heuser

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Negative Curvature ◽

Multivesicular Body ◽

Multivesicular Bodies ◽

Deficient Mutant ◽

Membrane Deformation ◽

Endosomal Sorting ◽

Viral Budding ◽

Circular Arrays

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used “deep-etch” electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis–deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.

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Structure and function of ESCRT-III

Biochemical Society Transactions ◽

10.1042/bst0370156 ◽

2009 ◽

Vol 37 (1) ◽

pp. 156-160 ◽

Cited By ~ 48

Author(s):

Suman Lata ◽

Guy Schoehn ◽

Julianna Solomons ◽

Ricardo Pires ◽

Heinrich G. Göttlinger ◽

...

Keyword(s):

Multivesicular Body ◽

Multivesicular Bodies ◽

Tubular Structures ◽

Body Protein ◽

Enveloped Viruses ◽

Endosomal Sorting ◽

Vacuolar Protein ◽

And Function ◽

Endosomal Vesicles

ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A–CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.

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The CHMP4b- and Src-docking sites in the Bro1 domain are autoinhibited in the native state of Alix

Biochemical Journal ◽

10.1042/bj20081388 ◽

2009 ◽

Vol 418 (2) ◽

pp. 277-284 ◽

Cited By ~ 22

Author(s):

Xi Zhou ◽

Shujuan Pan ◽

Le Sun ◽

Joe Corvera ◽

Yu-Chen Lee ◽

...

Keyword(s):

Multivesicular Body ◽

Human Embryonic Kidney ◽

Native State ◽

Interacting Protein ◽

Body Protein ◽

Linked Gene ◽

Endosomal Sorting ◽

Protein X ◽

Membrane Bound ◽

Docking Sites

The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.

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A dominant-negative ESCRT-III protein perturbs cytokinesis and trafficking to lysosomes

Biochemical Journal ◽

10.1042/bj20071296 ◽

2008 ◽

Vol 411 (2) ◽

pp. 233-239 ◽

Cited By ~ 29

Author(s):

Joseph D. Dukes ◽

Judith D. Richardson ◽

Ruth Simmons ◽

Paul Whitley

Keyword(s):

Membrane Trafficking ◽

Multivesicular Body ◽

Dominant Negative ◽

Eukaryotic Cells ◽

Body Protein ◽

Recycling Endosomes ◽

Protein Subunits ◽

Autoinhibitory Domain ◽

Endosomal Sorting ◽

A Subunit

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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MITD1 is recruited to midbodies by ESCRT-III and participates in cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e12-04-0292 ◽

2012 ◽

Vol 23 (22) ◽

pp. 4347-4361 ◽

Cited By ~ 17

Author(s):

Seongju Lee ◽

Jaerak Chang ◽

Benoît Renvoisé ◽

Anita Tipirneni ◽

Sarah Yang ◽

...

Keyword(s):

Critical Role ◽

Multivesicular Body ◽

Strong Interactions ◽

Body Protein ◽

Endosomal Sorting ◽

Cellular Processes ◽

Viral Budding ◽

Membrane Fission ◽

Downstream Effects ◽

Trafficking Molecules

Diverse cellular processes, including multivesicular body formation, cytokinesis, and viral budding, require the sequential functions of endosomal sorting complexes required for transport (ESCRTs) 0 to III. Of these multiprotein complexes, ESCRT-III in particular plays a key role in mediating membrane fission events by forming large, ring-like helical arrays. A number of proteins playing key effector roles, most notably the ATPase associated with diverse cellular activities protein VPS4, harbor present in microtubule-interacting and trafficking molecules (MIT) domains comprising asymmetric three-helical bundles, which interact with helical MIT-interacting motifs in ESCRT-III subunits. Here we assess comprehensively the ESCRT-III interactions of the MIT-domain family member MITD1 and identify strong interactions with charged multivesicular body protein 1B (CHMP1B), CHMP2A, and increased sodium tolerance-1 (IST1). We show that these ESCRT-III subunits are important for the recruitment of MITD1 to the midbody and that MITD1 participates in the abscission phase of cytokinesis. MITD1 also dimerizes through its C-terminal domain. Both types of interactions appear important for the role of MITD1 in negatively regulating the interaction of IST1 with VPS4. Because IST1 binding in turn regulates VPS4, MITD1 may function through downstream effects on the activity of VPS4, which plays a critical role in the processing and remodeling of ESCRT filaments in abscission.

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

10.1101/361659 ◽

2018 ◽

Author(s):

Jessica B.A. Sadler ◽

Dawn M. Wenzel ◽

Lauren K. Williams ◽

Marta Guindo-Martínez ◽

Steven L. Alam ◽

...

Keyword(s):

Dna Damage ◽

Cancer Susceptibility ◽

Genome Instability ◽

Multivesicular Body ◽

Mitotic Checkpoint ◽

Extensive Study ◽

Body Protein ◽

Endosomal Sorting ◽

Physiological Importance ◽

Mitotic Stress

AbstractCytokinetic 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 CHMP4CT232 (rs35094336), 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 mis-segregation, 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.Significance StatementThe final step of cell division, abscission, is temporally regulated by the Aurora B kinase and CHMP4C in a conserved pathway called the abscission checkpoint which arrests abscission in the presence of lingering mitotic problems. Despite extensive study, the physiological importance of this pathway to human health has remained elusive. We now demonstrate that a cancer predisposing polymorphism in CHMP4C disrupts the abscission checkpoint and results in DNA damage accumulation. Moreover, deficits in this checkpoint synergize with p53 loss and generate aneuploidy under stress conditions that increase the frequency of chromosome missegregation. Therefore, cells expressing the cancer-associated polymorphism in CHMP4C are genetically unstable, thus suggesting a novel oncogenic mechanism that may involve the dysregulation of abscission.

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The role of CHMP2B in frontotemporal dementia

Biochemical Society Transactions ◽

10.1042/bst0370208 ◽

2009 ◽

Vol 37 (1) ◽

pp. 208-212 ◽

Cited By ~ 41

Author(s):

Hazel Urwin ◽

Shabnam Ghazi-Noori ◽

John Collinge ◽

Adrian Isaacs

Keyword(s):

Frontotemporal Dementia ◽

Neuroblastoma Cells ◽

Multivesicular Body ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Endosomal Trafficking ◽

Human Neuroblastoma ◽

Body Protein ◽

Endosomal Sorting ◽

Negative Effect

Mutations in the CHMP2B (charged multivesicular body protein 2B) gene that lead to C-terminal truncations of the protein can cause frontotemporal dementia. CHMP2B is a member of ESCRT-III (endosomal sorting complex required for transport III), which is required for formation of the multivesicular body, a late endosomal structure that fuses with the lysosome to degrade endocytosed proteins. Overexpression of mutant C-terminally truncated CHMP2B proteins produces an enlarged endosomal phenotype in PC12 and human neuroblastoma cells, which is likely to be due to a dominant-negative effect on endosomal function. Disruption of normal endosomal trafficking is likely to affect the transport of neuronal growth factors and autophagic clearance of proteins, both of which could contribute to neurodegeneration in frontotemporal dementia.

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The ESCRTs – converging on mechanism

Journal of Cell Science ◽

10.1242/jcs.240333 ◽

2020 ◽

Vol 133 (18) ◽

pp. jcs240333 ◽

Cited By ~ 1

Author(s):

Mark Remec Pavlin ◽

James H. Hurley

Keyword(s):

Associated Factors ◽

Multivesicular Body ◽

Underlying Mechanism ◽

Complex Nature ◽

Mechanistic Studies ◽

Membrane Repair ◽

Helical Structures ◽

Endosomal Sorting ◽

Geometric Transitions

ABSTRACTThe endosomal sorting complexes required for transport (ESCRTs) I, -II and –III, and their associated factors are a collection of ∼20 proteins in yeast and ∼30 in mammals, responsible for severing membrane necks in processes that range from multivesicular body formation, HIV release and cytokinesis, to plasma and lysosomal membrane repair. ESCRTs are best known for ‘reverse-topology’ membrane scission, where they act on the inner surface of membrane necks, often when membranes are budded away from the cytosol. These events are driven by membrane-associated assemblies of dozens to hundreds of ESCRT molecules. ESCRT-III proteins form filaments with a variety of geometries and ESCRT-I has now been shown to also form helical structures. The complex nature of the system and the unusual topology of its action has made progress challenging, and led to controversies with regard to its underlying mechanism. This Review will focus on recent advances obtained by structural in vitro reconstitution and in silico mechanistic studies, and places them in their biological context. The field is converging towards a consensus on the broad outlines of a mechanism that is driven by a progressive ATP-dependent treadmilling exchange of ESCRT subunits, as well as compositional change and geometric transitions in ESCRT filaments.

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