Integrated Structural Model and Membrane Targeting Mechanism of the Human ESCRT-II Complex

Im, Young Jun; Hurley, James H.

doi:10.1016/j.devcel.2008.04.004

Cited by 92 publications

(155 citation statements)

References 45 publications

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“…Consistent with a role for ESCRT-II in recruiting ESCRT-III to cellular membranes [17][18][19], the ESCRT-II-like N terminus of CHMP7 directs ESCRT-III assembly at the NE. In C. elegans, ESCRT-II has been reported to localize to the sarcoplasmic reticulum, suggesting that the tandem WH fold may play a broader role in ER targeting [20].…”

Section: Resultsmentioning

confidence: 77%

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

Carlton

Perdrix

Olmos

2016

Preprint

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[6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleocytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration. RESULTS AND DISCUSSIONCHMP7 is unique among endosomal sorting complex required for transport-III (ESCRT-III) subunits in that it contains an extended N terminus (NT) (Figures 1A and S1A) that we hypothesized may be important during its role in nuclear envelope (NE) regeneration. Using a dual-nickase CRISPR/Cas9 approach [7], we edited the CHMP7 locus in CAL-51 cells to produce a homozygous N-terminal fusion of monomeric-NeonGreen (mNG) [8] to CHMP7 under the control of its endogenous promoter (Figures 1B and S1B-S1I). These cells grew normally, suggesting that N-terminal tagging of CHMP7 is benign. We imaged living mNG-CHMP7 cells and found that, while CHMP7 was recruited to the NE during mitotic exit, in addition to a cytoplasmic pool, it decorated ER membranes in interphase and mitotic cells (Figures 1C and S1J; Movie S1). Stably expressed GFP-CHMP7 localized similarly ( Figures 1D-1F and S1K-S1M). We saw no localization of GFP-CHMP7 to the midbody ( Figure S1N). CHMP7-antisera failed to detect small interfering RNA (siRNA)-sensitive immunofluorescence signal; however, we could detect endogenous CHMP7 in ER fractions from homogenized cells ( Figures 1G and S1O). S. cerevisiae Chm7 was recently shown to localize to the ER [6], suggesting that this localization is evolutionarily conserved. During NE reformation, all other ESCRT-III subunits are recruited from the cytoplasm [2,3]; given that the NE is formed from the ER [9, 10], a pre-existing ER localization for CHMP7 suggested a platform from which this recruitment could occur. Analysis of HeLa cells stably expressing GFP-CHMP7 NT or mCh-CHMP7 NT revealed that CHMP7's N terminus directed localization to the ER, but this truncated protein exhibited little stabilization at the reforming NE ( Figures 1D and S2A-S2D; Movie S2). In contrast, the C terminus of CHMP7 (GFP-CHMP7 dNT) was cytosolic and displayed neither ER localization nor stabilization at the reforming NE ( Figure 1H; Movie S2), despite containing the CHMP4B/ESCRT-III interaction domain [11]. CHMP7 is responsible for recruiting downstream ESCRT-III components to the reforming NE through CHMP4B. Fusion of siRNA-resistant CHMP7 NT to CHMP4B directed cytoplasmic CHMP4B to the mitotic ER and restored its enrichment at sites of annular fusion at the forming NE, in the absence of endogenous CHMP7 (Figures S2E-S2G).Analysis of the secondary structure of CHMP7 NT has revealed the presence of tandem winged helix (WH) domains [6,12], resembling those found in ESCRT-II subunits ( Figure S...

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Section: Resultsmentioning

confidence: 77%

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

Carlton

Perdrix

Olmos

2016

Preprint

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“…Membrane targeting via nonspecific binding to acidic lipids is most commonly carried out by polylysine tracts and basic amphipathic helices (38). Indeed, the ESCRT pathway itself provides some examples, including basic putative helices at the N termini of Vps37 of yeast ESCRT-I (10) and Vps22 of yeast and human ESCRT-II (39). It is intriguing that sometimes specialized domains, such as MABP and KA1, use well-defined three-dimensional folds to carry out the same function.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

Bouřa

Hurley

2012

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

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MVB12-associated β-prism (MABP) domains are predicted to occur in a diverse set of membrane-associated bacterial and eukaryotic proteins, but their existence, structure, and biochemical properties have not been characterized experimentally. Here, we find that the MABP domains of the MVB12A and B subunits of ESCRT-I are functional modules that bind in vitro to liposomes containing acidic lipids depending on negative charge density. The MABP domain is capable of autonomously localizing to subcellular puncta and to the plasma membrane. The 1.3-Å atomic resolution crystal structure of the MVB12B MABP domain reveals a β-prism fold, a hydrophobic membrane-anchoring loop, and an electropositive phosphoinositide-binding patch. The basic patch is open, which explains how it senses negative charge density but lacks stereoselectivity. These observations show how ESCRT-I could act as a coincidence detector for acidic phospholipids and protein ligands, enabling it to function both in protein transport at endosomes and in cytokinesis and viral budding at the plasma membrane.HIV | protein structure | protein-membrane interactions | X-ray crystallography | subcellular localization T he concept of modular membrane-targeting domains is an important theme in the architecture of regulatory proteins and central to our current understanding of the subcellular localization of proteins (1-3). The best known of these modules include the PH, PX, FYVE, C1, C2, and ENTH domains (1-3). Each of these domains was initially proposed on the basis of bioinformatics analysis and subsequently confirmed by structural, biochemical, and cell biological approaches.A recent bioinformatics analysis (4) led to the identification of a putative membrane-targeted domain, termed the "MVB12-associated β-prism" (MABP) domain (4). MABP domains are found at the N termini of MVB12A and B, which are subunits of the human ESCRT-I complex (5). ESCRT complexes are responsible for the endosomal sorting of ubiquitinated membrane proteins into multivesicular bodies (MVBs) en route to their lysosomal degradation (6, 7). ESCRT-I in particular is also involved in plasma membrane functions in cytokinesis and the release of HIV-1 and other viruses (8). MABP domains are also found in many membrane-associated bacterial proteins and in the N-terminal region of human DENND4 isoforms. DENND4 proteins are Rab10 guanine nucleotide exchange factors that localize to a tubular Golgi-proximal membrane compartment (9).We set out to test the inference that MABP domains interact with membranes and found that the MVB12A and B MABP domains did. The crystal structure, solved at atomic resolution, showed that the MABP domain has a β-prism fold, consistent with the bioinformatics analysis. The structure identified a potential membrane-binding site, which we confirmed experimentally in vitro and in vivo. We found that the domain bound to liposomes according to the negative charge density of the membrane but with little specificity for particular lipid head groups. We went on to characterize the...

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“…ESCRT‐I is a claviform heterotetramer of tumor susceptibility gene 101, vacuolar protein sorting‐associated protein 28 (VPS28), the VPS37 Homolog (VPS37A/B/C/D), and one of the multivesicular body subunit 12A (MVB12A), MVB12B, or ubiquitin‐associated protein 1 61, 62. ESCRT‐II is a bifurcate heterotetramer consisting of one molecule each of VPS22 and VPS36 and two molecules of VPS25 63, 64, 65. The VPS28 subunit of ESCRT‐I and the VPS36 subunit of ESCRT‐II participate in the connection of the two complexes 66, 67, 68.…”

Section: Biogenesis and Release Of Evsmentioning

confidence: 99%

Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine

2018

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Extracellular vesicles (EVs) are ubiquitous nanosized membrane vesicles consisting of a lipid bilayer enclosing proteins and nucleic acids, which are active in intercellular communications. EVs are increasingly seen as a vital component of many biological functions that were once considered to require the direct participation of stem cells. Consequently, transplantation of EVs is gradually becoming considered an alternative to stem cell transplantation due to their significant advantages, including their relatively low probability of neoplastic transformation and abnormal differentiation. However, as research has progressed, it is realized that EVs derived from native‐source cells may have various shortcomings, which can be corrected by modification and optimization. To date, attempts are made to modify or improve almost all the components of EVs, including the lipid bilayer, proteins, and nucleic acids, launching a new era of modularized EV therapy through the “modular design” of EV components. One high‐yield technique, generating EV mimetic nanovesicles, will help to make industrial production of modularized EVs a reality. These modularized EVs have highly customized “modular design” components related to biological function and targeted delivery and are proposed as a promising approach to achieve personalized and precision medicine.

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Integrated Structural Model and Membrane Targeting Mechanism of the Human ESCRT-II Complex

Cited by 92 publications

References 45 publications

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit

Modularized Extracellular Vesicles: The Dawn of Prospective Personalized and Precision Medicine

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