Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

Obita, Takayuki; Saksena, Suraj; Ghazi-Tabatabai, Sara; Gill, David; Perišić, Olga; Emr, Scott D.; Williams, Roger L.

doi:10.1038/nature06171

Cited by 290 publications

(443 citation statements)

References 30 publications

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“…Like the CHMP4 subunits, the CHMP1-3 subunits of ESCRT-III also have C-terminal amphipathic helices [MIT interacting motifs (MIM)], but in these cases the helices bind the MIT domains of VPS4, Vta1p/ LIP5, and AMSH and thereby recruit ATPase and deubiquitylating activities to the membrane (43)(44)(45)(46)(47)(48). Hence, the terminal helices of different ESCRT-III subunits must display distinct binding surfaces to ensure specificity in protein recruitment.…”

Section: Discussionmentioning

confidence: 99%

ALIX-CHMP4 interactions in the human ESCRT pathway

McCullough

Fisher

Whitby

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

214

252

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The ESCRT pathway facilitates membrane fission events during enveloped virus budding, multivesicular body formation, and cytokinesis. To promote HIV budding and cytokinesis, the ALIX protein must bind and recruit CHMP4 subunits of the ESCRT-III complex, which in turn participate in essential membrane remodeling functions. Here, we report that the Bro1 domain of ALIX binds specifically to C-terminal residues of the human CHMP4 proteins (CHMP4A-C). Crystal structures of the complexes reveal that the CHMP4 C-terminal peptides form amphipathic helices that bind across the conserved concave surface of ALIXBro1. ALIX-dependent HIV-1 budding is blocked by mutations in exposed ALIXBro1 residues that help contribute to the binding sites for three essential hydrophobic residues that are displayed on one side of the CHMP4 recognition helix (M/L/IxxLxxW). The homologous CHMP1-3 classes of ESCRT-III proteins also have C-terminal amphipathic helices, but, in those cases, the three hydrophobic residues are arrayed with L/I/MxxxLxxL spacing. Thus, the distinct patterns of hydrophobic residues provide a ''code'' that allows the different ESCRT-III subunits to bind different ESCRT pathway partners, with CHMP1-3 proteins binding MIT domain-containing proteins, such as VPS4 and Vta1/LIP5, and CHMP4 proteins binding Bro1 domaincontaining proteins, such as ALIX.cytokinesis ͉ ESCRT-III ͉ HIV ͉ mutivesicular body

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Section: Discussionmentioning

confidence: 99%

ALIX-CHMP4 interactions in the human ESCRT pathway

McCullough

Fisher

Whitby

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

214

252

View full text Add to dashboard Cite

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“…To answer this, we used a dominant-negative VPS2 mutant (VPS2-DN), which was generated by deleting the C-terminal MIT-interacting motif responsible for the interaction with SKD1 (Obita et al, 2007;Hurley and Yang, 2008). Vacuolar transport of the reporter a-amylase-sporamin (amy-spo) was measured as the secretion index (SI) given by the ratio of amy-spo detected in the culture medium and within the cells (Pimpl et al, 2003).…”

Section: Vps2-dn and Treatment With Conca Prevent The Arrival Of Solumentioning

confidence: 99%

Multivesicular Bodies Mature from the Trans-Golgi Network/Early Endosome in Arabidopsis

et al. 2011

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The plant trans-Golgi network/early endosome (TGN/EE) is a major hub for secretory and endocytic trafficking with complex molecular mechanisms controlling sorting and transport of cargo. Vacuolar transport from the TGN/EE to multivesicular bodies/late endosomes (MVBs/LEs) is assumed to occur via clathrin-coated vesicles, although direct proof for their participation is missing. Here, we present evidence that post-TGN transport toward lytic vacuoles occurs independently of clathrin and that MVBs/LEs are derived from the TGN/EE through maturation. We show that the V-ATPase inhibitor concanamycin A significantly reduces the number of MVBs and causes TGN and MVB markers to colocalize in Arabidopsis thaliana roots. Ultrastructural analysis reveals the formation of MVBs from the TGN/EE and their fusion with the vacuole. The localization of the ESCRT components VPS28, VPS22, and VPS2 at the TGN/EE and MVBs/LEs indicates that the formation of intraluminal vesicles starts already at the TGN/EE. Accordingly, a dominant-negative mutant of VPS2 causes TGN and MVB markers to colocalize and blocks vacuolar transport. RNA interference-mediated knockdown of the annexin ANNAT3 also yields the same phenotype. Together, these data indicate that MVBs originate from the TGN/EE in a process that requires the action of ESCRT for the formation of intraluminal vesicles and annexins for the final step of releasing MVBs as a transport carrier to the vacuole.

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“…To generate DN versions of the CHMPs (CHMP-DN) they were cloned as yellow fluorescent protein (YFP) fusion proteins such that the YFP moiety was C-terminal (Pawliczek & Crump, 2009), this prevents the C terminus-mediated interaction of the CHMPs with Vps4 (Obita et al, 2007;Stuchell-Brereton et al, 2007) and therefore perturbs ESCRT complex recycling. These constructs were then introduced into the trans-complementation system, as described above, to determine if they were able to inhibit HCV particle production.…”

Section: Inhibition Of Vps4 Function Blocks Hcv Particle Productionmentioning

confidence: 99%

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Corless

Crump

Griffin

et al. 2009

Journal of General Virology

102

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The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving transcomplementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focusforming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles. INTRODUCTIONHepatitis C virus (HCV) represents a major global health burden. An estimated 170 million people are chronically infected worldwide and a significant proportion of these will go on to develop end stage liver disease. Current drug treatments are poorly tolerated and are only effective in approximately 50 % of individuals. There is, therefore, a pressing need for a greater understanding of the molecular mechanisms involved in HCV infection in order for novel drug targets to be identified.HCV is the prototype member of the genus Hepacivirus in the family Flaviviridae. It is a single-stranded, positive-sense RNA virus whose genome encodes a single ORF. This is translated in a cap-independent manner from an internal ribosome entry site (IRES) located in the 59 untranslated region, into a single polyprotein of approximately 3000 aa. The polyprotein is proteolytically cleaved by host and viral proteases to form 10 mature proteins; the structural proteins (core, E1 and E2), a viroporin p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).Although recent advances in the development of tissue culture systems for the study of the HCV life cycle (Lindenbach et al., 2005;Wakita et al., 2005;Zhong et al., 2005) have defined a critical role for lipid droplets in virus assembly (Miyanari et al., 2007), the precise mechanism by which infectious HCV particles are assembled and released remains poorly characterized. In particular, it has not been established how nascent...

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Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

Cited by 290 publications

References 30 publications

ALIX-CHMP4 interactions in the human ESCRT pathway

ALIX-CHMP4 interactions in the human ESCRT pathway

Multivesicular Bodies Mature from the Trans-Golgi Network/Early Endosome in Arabidopsis

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

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