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

2009 ◽  
Vol 418 (2) ◽  
pp. 277-284 ◽  
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.

The ESCRT pathway and HIV-1 budding

2009 ◽  
Vol 37 (1) ◽  
pp. 181-184 ◽  
Author(s):  
Yoshiko Usami ◽  
Sergei Popov ◽  
Elena Popova ◽  
Michio Inoue ◽  
Winfried Weissenhorn ◽  
...  
Keyword(s):  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Intramolecular Interactions ◽  
Transport Pathway ◽  
Interacting Protein ◽  
Linked Gene ◽  
Endosomal Sorting ◽  
Binding Partner ◽  
Protein X ◽  
Hiv 1

HIV-1 Gag engages components of the ESCRT (endosomal sorting complex required for transport) pathway via so-called L (late-assembly) domains to promote virus budding. Specifically, the PTAP (Pro-Thr-Ala-Pro)-type primary L domain of HIV-1 recruits ESCRT-I by binding to Tsg101 (tumour susceptibility gene 101), and an auxiliary LYPXnL (Leu-Tyr-Pro-Xaan-Leu)-type L domain recruits the ESCRT-III-binding partner Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X]. The structurally related CHMPs (charged multivesicular body proteins), which form ESCRT-III, are kept in an inactive state through intramolecular interactions, and become potent inhibitors of HIV-1 budding upon removal of an autoinhibitory region. In the absence of the primary L domain, HIV-1 budding is strongly impaired, but can be efficiently rescued through the overexpression of Alix. This effect of Alix depends on its ability to interact with CHMP4, suggesting that it is the recruitment of CHMPs that ultimately drives virus release. Surprisingly, HIV-1 budding defects can also be efficiently corrected by overexpressing Nedd (neural-precursor-cell-expressed developmentally down-regulated) 4-2s, a member of a family of ubiquitin ligases previously implicated in the function of PPXY (Pro-Pro-Xaa-Tyr)-type L domains, which are absent from HIV-1. At least under certain circumstances, Nedd4-2s stimulates the activity of PTAP-type L domains, raising the possibility that the ubiquitin ligase regulates the activity of ESCRT-I.

The HIV-1 p6/EIAV p9 docking site in Alix is autoinhibited as revealed by a conformation-sensitive anti-Alix monoclonal antibody

2008 ◽  
Vol 414 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Xi Zhou ◽  
Shujuan Pan ◽  
Le Sun ◽  
Joe Corvera ◽  
Sue-Hwa Lin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Three Dimensional ◽  
Docking Site ◽  
Positive Role ◽  
Interacting Protein ◽  
Linked Gene ◽  
Endosomal Sorting ◽  
Protein X ◽  
Equine Infectious Anaemia Virus ◽  
Hiv 1

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6Gag or EIAV (equine infectious anaemia virus) p9Gag, and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6Gag/p9Gag and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active p6Gag/p9Gag docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6Gag/p9Gag docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.

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.

Dengue virus requires apoptosis linked gene-2-interacting protein X (ALIX) for viral propagation

2019 ◽  
Vol 261 ◽  
pp. 65-71 ◽  
Author(s):  
Chutima Thepparit ◽  
Sarawut Khongwichit ◽  
Kunjimas Ketsuwan ◽  
Sirikwan Libsittikul ◽  
Prasert Auewarakul ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Interacting Protein ◽  
Linked Gene ◽  
Viral Propagation ◽  
Protein X

Early increase of apoptosis-linked gene-2 interacting protein X in areas of kainate-induced neurodegeneration

Neuroscience ◽  
2004 ◽  
Vol 123 (4) ◽  
pp. 887-895 ◽  
Author(s):  
F.J Hemming ◽  
S Fraboulet ◽  
B Blot ◽  
R Sadoul
Keyword(s):  
Interacting Protein ◽  
Linked Gene ◽  
Protein X ◽  
Early Increase

Increased Alix (apoptosis-linked gene-2 interacting protein X) immunoreactivity in the degenerating striatum of rats chronically treated by 3-nitropropionic acid

2004 ◽  
Vol 368 (3) ◽  
pp. 309-313 ◽  
Author(s):  
David Blum ◽  
Fiona J. Hemming ◽  
Marie-Christine Galas ◽  
Sakina Torch ◽  
Laetitia Cuvelier ◽  
...  
Keyword(s):  
Interacting Protein ◽  
Linked Gene ◽  
Nitropropionic Acid ◽  
Protein X ◽  
3 Nitropropionic Acid

Structure and function of ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 156-160 ◽  
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.

scholarly journals Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

2020 ◽  
Author(s):  
Moona Huttunen ◽  
Artur Yakimovich ◽  
Ian J. White ◽  
Janos Kriston-Vizi ◽  
Juan Martin-Serrano ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Multivesicular Body ◽  
Cellular Membrane ◽  
Double Membrane ◽  
Endosomal Sorting ◽  
A Cell ◽  
Infected Cells ◽  
Membrane Bound ◽  
Virus Egress

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

Peroxisome biogenesis and the formation of multivesicular peroxisomes during tombusvirus infection: a role for ESCRT?This review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 551-564 ◽  
Author(s):  
Robert T. Mullen ◽  
Andrew W. McCartney ◽  
C. Robb Flynn ◽  
Graham S.T. Smith
Keyword(s):  
Cell Biology ◽  
Molecular Mechanisms ◽  
Protein Sorting ◽  
Multivesicular Body ◽  
Peroxisomal Membrane ◽  
Viral Pathogens ◽  
Endosomal Sorting ◽  
Metabolic Functions ◽  
Membrane Bound ◽  
Vacuolar Protein

Peroxisomes are highly dynamic organelles with regard to their metabolic functions, shapes, distribution, movements, and biogenesis. They are also important as sites for the development of some viral pathogens. It has long been known that certain members of the tombusvirus family recruit peroxisomes for viral RNA replication and that this process is accompanied by dramatic changes in peroxisome morphology, the most remarkable of which is the extensive inward vesiculation of the peroxisomal boundary membrane leading to the formation of a peroxisomal multivesicular body (pMVB). While it is unclear how the internal vesicles of a pMVB form, they appear to serve in effectively concentrating viral membrane-bound replication complexes and protecting nascent viral RNAs from host-cell defences. Here, we review briefly the biogenesis of peroxisomes and pMVBs and discuss recent studies that have begun to shed light on how components of the tombusvirus replicase exploit the molecular mechanisms involved in peroxisome membrane protein sorting. We also address the question of what controls invagination and vesicle formation at the peroxisomal membrane during pMVB biogenesis. We propose that tombusviruses exploit protein constituents of the class E vacuolar protein-sorting pathway referred to as ESCRT (endosomal sorting complex required for transport) in the formation of pMVBs. This new pMVB–ESCRT hypothesis reconciles current paradigms of pMVB biogenesis with the role of ESCRT in endosomal multivesicular body formation and the ability of enveloped RNA viruses, including HIV, to appropriate the ESCRT machinery to execute their budding programme from cells.

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