MEMBRANE REMODELING

Structure and membrane remodelingactivity of ESCRT-III helical polymersJohn McCullough,1* Amy K. Clippinger,2* Nathaniel Talledge,1,3 Michael L. Skowyra,2

Marissa G. Saunders,1 Teresa V. Naismith,2 Leremy A. Colf,1 Pavel Afonine,4

Christopher Arthur,5 Wesley I. Sundquist,1† Phyllis I. Hanson,2† Adam Frost1,3†

The endosomal sorting complexes required for transport (ESCRT) proteins mediatefundamental membrane remodeling events that require stabilizing negative membranecurvature. These include endosomal intralumenal vesicle formation, HIV budding,nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits performkey roles in these processes by changing conformation and polymerizing intomembrane-remodeling filaments. Here, we report the 4 angstrom resolutioncryogenic electron microscopy reconstruction of a one-start, double-strandedhelical copolymer composed of two different human ESCRT-III subunits, chargedmultivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1).The inner strand comprises “open” CHMP1B subunits that interlock in an elaboratedomain-swapped architecture and is encircled by an outer strand of “closed” IST1subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form externalcoats on positively curved membranes in vitro and in vivo. Our analysis suggests howcommon ESCRT-III filament architectures could stabilize different degrees anddirections of membrane curvature.

The endosomal sorting complexes requiredfor transport (ESCRT) pathway is best knownfor facilitating membrane remodeling andfission for processes such as the endosomalintralumenal vesicle (ILV) formation, envel-

oped virus budding, nuclear envelope closure,and cytokinetic abscission (1–3). In these reac-tions, the ESCRT machinery assembles on theinterior of a negatively curved, cytoplasm-filledmembrane neck and pulls themembrane towarditself to the fission point. These fission reactionsare topologically distinct from reactions inwhichcytoplasmic BAR domain–containing proteinsand dynamin-family guanosine triphosphatasesassemble around and constrict positively curvedmembrane tubules.ESCRT components are recruited to different

membranes by site-specific adaptors that ultimate-ly recruit ESCRT-III subunits and their bindingpartners, including VPS4-family adenosine tri-phosphatases (ATPases). ESCRT-III assembliespromote membrane constriction and fission, pos-sibly in concert with VPS4. Humans express 12related ESCRT-III proteins, called chargedmulti-vesicular bodyproteins (CHMPs) 1A-7 and increasedsodium tolerance 1 (IST1) (1–3). Crystal structuresof CHMP3 and IST1 show a common structure in

which the first two helices form a long hairpin,the shorter helices 3 and 4 pack against the openend of the hairpin, and helix 5 folds back andpacks against the closed end of the helical hair-pin (4–6). This “closed” conformation appears toautoinhibit ESCRT-III membrane binding andoligomerization (4, 7, 8). ESCRT-III subunits canalso adopt a second, more extended “open” con-formation that has been characterized biochem-ically, but not visualized in molecular detail.The open conformation appears to be the active,polymerization-competent state because mutationsor solution conditions that favor this conforma-tion typically promote polymerization (1–3).ManyESCRT-III subunits form spiraling homo- andheteromeric filaments, both in vitro (4, 9–15) andin cells (10, 16–19), but the structural basis forfilament assembly is unclear.We used cryogenic electron microscopy (cryo-

EM) to determine the molecular structure of ahelical copolymer comprising human IST1 andCHMP1B. Full-length IST1 and CHMP1B spon-taneously coassembled under low ionic strengthconditions into well-ordered helical tubes. Helicalorder was further enhanced by using a truncatedIST1 construct that spanned residues 1 to 189,hereafter termed IST1NTD, and by including small,acidic unilamellar vesicles (SUVs) to nucleate poly-mer formation. The resulting IST1NTD-CHMP1Btubes were long, straight, and 24 nm in diameter(Fig. 1A). The three-dimensional (3D) structureof IST1NTD-CHMP1B assemblies was determinedto a resolution of ~4 Å by real-space helical re-construction (supplementary materials, materialsand methods, and figs. S1 to S4). Each tubecomprised a right-handed one-start helical fila-ment that packed with an interfilament spacingof 5.1 nm per turn (Fig. 1, B to D, and movie S1).

Each filamentwas double-stranded,with distinctinner and outer strands (at 7.7 and 10.2 nm radii,respectively). Segmented densities from sub-units in the outer strand correspondedwell to thecrystal structure of IST1NTD in its closed confor-mation [Protein Data Bank (PDB) 3FRR] (5), withonly minor refinements required to optimize theposition of helix A (Fig. 1E and movie S2). Incontrast, the CHMP1B subunits of the innerstrand adopted a very different, open conforma-tion. These subunitswere almost entirelya-helical,and side chain densities were clearly evident inthe EM density (Fig. 1, B, D, and F; and movieS3). The open CHMP1B conformation resembledan arm, with helices 1 to 3 forming the upperarm and biceps, helix 4 and helix A forming theforearm, and helix 5 forming the hand. Jointsbetween helices 3 and 4 and between helices Aand 5 correspond to the elbow and wrist, respec-tively (Fig. 1, F and G).High-ionic-strength conditions (4, 8) typically

favor the monomeric, closed ESCRT-III subunitconformation (4, 8, 20, 21), and CHMP1B alsoremained monomeric under high-ionic-strengthconditions (fig. S5). Lowering the ionic strengthtriggered coassembly of IST1 and CHMP1B, im-plying that CHMP1B subunits are captured asthey open. To visualize this conformational change,we generated a structure-based homology modelfor the CHMP1B closed state (supplementary ma-terials, materials and methods). In comparisonwith themodeled closed state, the helix 5 hand isdisplaced by ~100 Å when CHMP1B opens. Thisglobal reorganization requires only three localrearrangements: The elbow angle between helices3 and 4 must change, and the loops that connecthelix 2/3 and helix 4/A must become helical tocreate the longer, continuous helices that extendthe upper arm to the elbow and create the fore-arm in the open state (Figs. 1, F and G, and 2Aand movie S4).In the filament, the open CHMP1B conforma-

tion is stabilized by extensive intersubunit inter-actions along the inner strand (Fig. 2B). EachCHMP1B molecule interacts with four otherCHMP1B subunits that pack together and crossthe forearm of the original subunit. In addition,the helix 5 hand grasps the shoulder of the hair-pin four subunits away,making adomain-swappedcontact that is analogous to the intrasubunitinteraction between the hairpin and helix 5in the closed ESCRT-III conformations (Fig.2, A and B). Opening and assembly reducesthe total solvent-accessible surface area ofCHMP1Bfrom ~10,720 to ~6,350 Å2. The IST1NTD-CHMP1Bassembly is further stabilized by three additionaltypes of interactions, which differ completelyfrom crystallized contacts for soluble IST1-CHMP1B heterodimers (Fig. 2, C to F, and figs.S6 and S7) (6).A final notable feature of the IST1-CHMP1B

tube is the remarkably cationic interior. This sur-face is created by a series of conserved basic resi-dues from helix 1 of CHMP1B (Fig. 2, D and F),which forms the principal membrane-bindingsite in other ESCRT-III proteins (5, 22). Its posi-tion inside the IST1-CHMP1B copolymer was

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1Department of Biochemistry, University of Utah, Salt LakeCity, UT 84112, USA. 2Department of Cell Biology andPhysiology, Washington University School of Medicine, St.Louis, MO 63110, USA. 3Department of Biochemistry andBiophysics, University of California, San Francisco, SanFrancisco, CA 94158, USA. 4Physical Bioscience Division,Lawrence Berkeley National Laboratory, Berkeley, CA 94720,USA. 5FEI Company, Hillsboro, OR 97124, USA.*These authors contributed equally to this work. †Correspondingauthor. E-mail: wes@biochem.utah.edu (W.I.S.); phanson22@wustl.edu (P.I.H.); adam.frost@ucsf.edu (A.F.)

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unexpected because well-characterized ESCRT-mediated membrane remodeling events requirethatmembranes interact with the exterior surface

of coiled ESCRT-III filaments, such as in the neckof a nascent ILV or viruses. The functional roles ofIST1 and CHMP1B in such canonical ESCRT

activities have been enigmatic, however, becauseneither protein is required for ILV biogenesis(23–26) or virus budding (27). Moreover, IST1

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Closed CHMP1B

Open CHMP1B

0 50 100 150 200

Closed IST1NTDα1 α2 α3 α4 αA αB α5 α6

Fig. 1. IST1NTD and CHMP1B copolymerized into helical tubes compris-ing polar, double-stranded helical filaments. (A) Electron cryomicrographshowing IST1NTD-CHMP1B tubes (white arrows) assembled by incubating equi-molar IST1NTD and CHMP1B in the presence of polymer-nucleating smallacidic unilamellar vesicles (SUVs; yellow arrows). (Inset) End-on viewof a shortIST1NTD-CHMP1B tube. Scale bars, 40 nm (A) and 20 nm (inset). (B) End-onviewof the reconstructed IST1NTD-CHMP1B tube highlighting single subunits of

IST1NTD (light green, outer strand) and CHMP1B (dark green, inner strand).(C) External view of the reconstructed helix with a highlighted IST1NTDsubunit. (D) Internal cutaway viewof the reconstructed helix,with a highlightedCHMP1B subunit. (E) Ribbon diagram of the modeled IST1NTD subunit (closedconformation). (F) Ribbon diagram of the modeled CHMP1B subunit (openconformation). (G) Secondary structure diagrams for closed IST1NTD (top),open CHMP1B (middle), and closed CHMP1B (bottom).

Closed

Open

i+1i+2i+3i+4 i

-10

kT/e

+200

Fig. 2. CHMP1B opening, strand structure, and electrostatic surface po-tentials of the IST1NTD-CHMP1B assembly. (A) Superposition of the openandclosedCHMP1Bconformations. (B) Five interlockedCHMP1Bmolecules fromthe inner strand of the filament. (C) “Top-end,” electrostatic surface view of theIST1NTD-CHMP1B tube, highlighting the acidity of the CHMP1B inner strand(including Glu130, Asp131, Asp147,Glu152, Asp155,Glu156, and Asp160) and the IST1NTDouter strand (includingAsp49,Glu50,Glu57,Glu163,Glu168,Glu178,Asp180, andGlu186).(D) “Bottom-end,” electrostatic surface view of the IST1NTD-CHMP1B tube,

highlighting the strongly basic characters of the CHMP1B inner strand (includingLys3, Lys87, Lys94, Lys101, Lys107, and Lys114) and the IST1NTD outer strand (includingLys7, Arg10, Lys90, Arg109, Lys118, Lys127, Lys130, Lys134, and Arg137). (E) Exterior,electrostatic surface view of the IST1NTD-CHMP1B tube, revealing the modestlybasic character of the IST1NTD outer strand. (F) Internal cutaway electrostaticsurface view of the IST1NTD-CHMP1B tube, revealing the strongly basic characterof the lumenal surface, contributed primarily by basic residues in CHMP1B helix1 (arrows), including Lys3, Lys13, Arg17, Lys20, Lys21, Lys24, Lys32, and Lys35.

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functions in the resolution of endosomal tu-bules that project into the cytoplasm and recyclecargoes back to the plasma membrane (28).Looking in cells, we found that moderately over-expressed CHMP1B polymerized into elongatedcytoplasmic structures (fig. S8). Deep-etch EM ofthe plasma membranes revealed filaments thatwere similar to those of CHMP4A and other well-studiedESCRT-IIIproteins (17), except thatCHMP1Bfilaments coated tubules ~35 to 60 nm in diam-eter that extended into rather than away fromthe cytoplasm (Fig. 3, A to D) (17, 18). Replicas ofcells transfected and immunolabeled for CHMP1Balone, or with IST1, also revealed immunodecor-ated organelles and tubules thatwere not attachedto the plasma membrane but again had recogniz-able striations (Fig. 3, E to K). The resemblance ofthese organelles to early endosomes (29), includ-ing the occasional presence of clathrin-coatedbuds (Fig. 3, J and K), indicates that CHMP1Band IST1 can coat and potentially remodel endo-somal tubules.To determine whether distinct membrane re-

modeling topologies are intrinsic properties ofdifferent ESCRT-III filaments, we compared thestructures induced by spirals of CHMP1B withthose induced by the prototypical ESCRT-III pro-tein CHMP4A. In earlier studies, CHMP4 pro-teins only deformed the membrane when boundto ATPase-deficient VPS4B (17) or an activatedCHMP2A mutant (18). We found that deletingC-terminal sequences yielded amutant CHMP4Athat formed tight, membrane-deforming spirals(Fig. 4A). The deformations induced by CHMP4Aspirals were directed away from the cytoplasm,as expected (17, 18). Comparable views of cellstransfected with full-length and C-terminal trun-cations of CHMP1B confirmed that CHMP4A andCHMP1B induced cellularmembranes to tubulatein opposite directions (Fig. 4B and fig. S9).We also tested how CHMP1B polymers bind

and remodel liposomal membranes in vitro. Un-der physiological solution conditions, CHMP1Bformed single- and double-stranded one-starthelices and spirals around membrane tubules,with interstrand spacing of 4.7 ± 0.1 nm (Fig. 4Cand figs. S10 and S11). Adding IST1 to CHMP1B-induced membrane tubules generated copoly-meric helices that were structural analogs ofthe membrane-free IST1-CHMP1B assembliesdescribed above, as judged by their interstrandspacing (5.1 ± 0.1 nm versus 5.2 ± 0.1 nm) andby the similarities of 2D class averages of the twoassemblies (Fig. 4D and figs. S10 and S11). Thepositively curved membrane tubules within theprotein coats could be visualized with negativestaining of the CHMP1B and IST1-CHMP1B as-semblies (Fig. 4, C and D, and fig. S10), in cryo-EM images, and in 2D class averages of themolecules along the tangential surface of thebilayer (fig. S11). Thus, CHMP1B and IST1 formexternal coats on positively curved membranesin vitro and in cells.Our analyses also provide insight into how

ESCRT-III filaments can assemble conical spiralsof decreasing diameter, as might be required todraw membranes together to the fission point.

In addition to uniform 24-nm helices, we fre-quently observed conical spirals of membrane-associated CHMP1B and IST1NTD-CHMP1B (Fig.4, C and D, and figs. S10 and S11), as well asconical IST1-CHMP1B filaments assembled inthe absence of nucleating vesicles (fig. S12).One class of membrane-free IST1NTD-CHMP1Bcones was sufficiently common to be recon-structed at low resolution, which confirmedthat the cones are composed of the same double-stranded filaments as those seen in the IST1NTD-CHMP1B helices (Fig. 4D; figs. S1, S11, and S12;andmovies S5 and S6). Within the conical spiral,both the degree of filament curvature and the

lateral interactions between adjacent filamentsvaried continuously. Small changes in filamentcurvature are likely accommodated by alteringthe angles of the “elbow” and “wrist” joints. Larg-er changes could, in principle, be accommodated(or even driven) by ratcheting the buttressingintersubunit interactions made by the “forearm”and “hand.” For example, changing the connec-tivity from i+4 to i+5would tend to straighten theCHMP1B filament. The required continuum ofdiffering lateral interactions in the IST1NTD-CHMP1Bspirals is apparently accommodated by smallshifts in ionic interstrand interactions, which isplausible because the basic charges are distributed

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Fig. 3. CHMP1B and IST1 tubulated cellular membranes. (A) Survey view of the cytoplasmic surfaceof the plasma membrane in an unroofed COS-7 cell expressing FLAG-CHMP1B. Tubular invaginationsextending into the cell interior are apparent along the exposed plasma membrane and as stabilizedopenings at the edges of the cell. Use view glasses for 3D viewing of anaglyphs (left eye, red). (B) Higher-magnification view of tubular invaginations induced and coated by FLAG-CHMP1B filaments. (C) Immuno-decoration confirmed the presence of CHMP1B around and along a tubule in a cell expressing untaggedCHMP1B. Antibody detected with 12 nm gold is white in these contrast-reversed EM images. (D) Higher-magnification view of FLAG-CHMP1B filament spirals on exposed plasma membrane. (E) CHMP1B-immunoreactive organelle in an unroofed COS-7 cell expressing untagged CHMP1B. Antibody detectedwith 12 nm gold is white in these contrast reversed EM images; a representative gold particle is circled inblue. (F to I) Representative internal tubules from cells coexpressing untagged CHMP1B (12 nm gold;example circled in blue) and IST1-myc (18 nm gold; examples circled in red). (J andK) Clathrin-coated budcapping the end of (J) CHMP1B and (K) IST1-myc immunolabeled tubules from cotransfected cells.Measurements of filament diameter (and interstrand spacing) showed that when apparently unitaryfilaments were resolvable, their diameter varied from 5 to 10 nm, including platinum.Thesemeasurementsare generally consistent with the dimensions of IST1-CHMP1B and CHMP1B filaments formed in vitro.Scale bars, 500 nm (A) and 100 nm (B) to (K).

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almost uniformly along one edge of the doublestrand (Fig. 2, C and D). Wider IST1-CHMP1Bspirals will tend to propagate toward theirpreferred 24-nm diameter. At this diameter, thenarrow lumen (~10 nm) would force internalopposing lipid bilayers (each ~4.7 nm wide) to-ward hemi-fission (30, 31), potentially providinga driving force for membrane constriction andfission. Last, we note that other ESCRT-III sub-units, such as CHMP4A, tubulate membranes inthe opposite direction to that seen with CHMP1B.Such stabilization of negative, rather than posi-tive, membrane curvature could be achievedsimply by altering the intrinsic degree and di-rection of filament curvature while retaining ananalogous membrane-binding surface and sub-unit connectivity.

REFERENCES AND NOTES

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389–402 (2008).18. A. G. Cashikar et al., eLife 3, e02184 (2014).19. J. Guizetti et al., Science 331, 1616–1620 (2011).20. A. Zamborlini et al., Proc. Natl. Acad. Sci. U.S.A. 103,

19140–19145 (2006).21. S. Shim, L. A. Kimpler, P. I. Hanson, Traffic 8, 1068–1079 (2007).22. N. J. Buchkovich, W. M. Henne, S. Tang, S. D. Emr, Dev. Cell

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(2006).26. S. M. Rue, S. Mattei, S. Saksena, S. D. Emr, Mol. Biol. Cell 19,

475–484 (2008).27. E. Morita, FEBS J. 279, 1399–1406 (2012).28. R. Allison et al., J. Cell Biol. 202, 527–543 (2013).29. W. Stoorvogel, V. Oorschot, H. J. Geuze, J. Cell Biol. 132,

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materials on Science Online.

ACKNOWLEDGMENTS

Electron microscopy was performed at the University of California,San Francisco, the University of Utah, and the Oregon Health &Science University (OHSU) Multiscale Microscopy Core withtechnical support from the FEI Living Lab and the OHSU Center forSpatial Systems Biomedicine (OCSSB). We thank B. Carragher,C. Potter, and the National Resource for Automated MolecularMicroscopy—which is supported by the National Institutes ofHealth (NIH) through the National Center for Research Resources(NCRR) P41 program (RR17573)—where some initial imagingstudies were performed. We also thank B. Bammes and DirectElectron, LP (San Diego, CA), W. Chiu, and J. Jakana at theNational Center for Macromolecular Imaging at Baylor College ofMedicine (Houston, Texas), where other images were collected.Computing support was provided by the Center for HighPerformance Computing at the University of Utah and the NSFExtreme Science and Engineering Development Environmentconsortium. We also thank C. Hill, F. Whitby, H. Schubert, B.Barad, and J. Fraser for guidance regarding model refinement andvalidation; D. Eckert for performing analytical ultracentrifugationexperiments; J. Iwasa for artwork; M. LaLonde, C. Rodesch,G. Mercenne, and M. Gudheti for help with supportingexperiments; and T. Goddard for Chimera 2 developments. Wethank R. Roth for deep-etch EM replicas, J. Heuser for sharingdeep-etch EM with us and advice, E. Clipperton for CHMP4Amutants, M. Pretz for IST1-myc constructs, and M. Hartstein forartwork. We thank K. Blumer, M. Babst, and other membersof our laboratories and colleagues for helpful discussions. Thisresearch was supported by the Searle Scholars Program(A.F.); the Jane Coffin Childs Foundation (M.G.S.); the PhenixIndustrial Consortium (P.A.); American Cancer Society grantPF-11-279-01-CCG (L.A.C.); U.S. Department of Energy contractDE-AC02-05CH11231 (P.A.); NIH grants 2P50GM082545-06(A.F.), 1DP2GM110772-01 (A.F.), R01GM112080 (W.I.S. and A.F.),R01AI051174 (W.I.S.), 1P01 GM063210 (P.A.), R01GM076686(P.I.H.), and R01NS050717 (P.I.H.); and NSF fellowshipDGE-1143954 (A.K.C.). All of the expression constructs used inour study have been or will be deposited in the public DNASUplasmid repository (http://dnasu.org/DNASU/Home.jsp).The density map has been deposited in the Electron MicroscopyData Bank (EMD-6461), and the fitted models were depositedin the PDB (3JC1).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/350/6267/1548/suppl/DC1Materials and MethodsFigs. S1 to S12Tables S1 and S2References (32–70)Movies S1 to S6

14 September 2015; accepted 16 November 2015Published online 3 December 201510.1126/science.aad8305

SCIENCE sciencemag.org 18 DECEMBER 2015 • VOL 350 ISSUE 6267 1551

CHMP4AInternal coat on

negatively-curved

membrane

CHMP1BExternal coat on

positively-curved

membrane

CHMP4A

CHMP1B

Cytoplasm

Cytoplasm

CHMP1B

CHMP1B + IST1NTD

Fig. 4. Topology of ESCRT-III membrane deformation in cells and in vitro. (A) Series of filamentspirals on the plasma membrane of COS-7 cells expressing CHMP4A1-164 show development of theoutwardly directed protrusions previously associated with ESCRT-III filaments (15, 16). Drawing high-lights relationship between a CHMP4A conical spiral and a negatively curved plasma membrane tubule.(B) Series of filament spirals on the plasma membrane of COS-7 cells expressing FLAG-CHMP1B showdevelopment of invaginations directed into the cell. Drawing highlights relationship between a CHMP1Bconical spiral and a positively curved plasma membrane tubule. (C) Negative stain electron micrographshowing that CHMP1B tubulates liposomes and forms a filamentous coat on the outside of the tubule.White arrows highlight regions coated by the CHMP1B helices, and the yellow arrow highlights a break inthe coat where the internal lipid is visible. (D) Negative stain electron micrograph showing that theIST1NTD-CHMP1B copolymer forms on the outside of membrane tubules.White arrows highlight regionscoated by the IST1NTD-CHMP1B copolymer, and the yellow arrows highlight breaks in the helical coat oruncoated regions of the liposome where the internal membrane is visible. Scale bars, 100 nm (A) and(B), 50 nm (C) and (D).

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Structure and membrane remodeling activity of ESCRT-III helical polymers

Leremy A. Colf, Pavel Afonine, Christopher Arthur, Wesley I. Sundquist, Phyllis I. Hanson and Adam FrostJohn McCullough, Amy K. Clippinger, Nathaniel Talledge, Michael L. Skowyra, Marissa G. Saunders, Teresa V. Naismith,

originally published online December 3, 2015DOI: 10.1126/science.aad8305 (6267), 1548-1551.350Science 

, this issue p. 1548Scienceturn ESCRT function on its head.mediate the budding of tubules and vesicles into the cytosol. Relatively minor structural rearrangements were required toopposite topology. ESCRT-III and IST1 ESCRT subunits form spirals on the outside of membrane tubules and so can

now show that ESCRTs can also promote the scission of membrane tubules with the completelyet al.McCullough budding. These reactions involve the formation of a cytoplasm-filled neck that spirals of the ESCRTs help to seal.

The so-called ESCRT proteins are involved in the budding of vesicles into the lumen of endosomes and in virusESCRTs work in two very different ways

ARTICLE TOOLS http://science.sciencemag.org/content/350/6267/1548

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2015/12/02/science.aad8305.DC1

REFERENCES

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