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1.
J Virol ; 98(2): e0190023, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38289107

RESUMO

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Interações entre Hospedeiro e Microrganismos , Nucleopoliedrovírus , Spodoptera , Proteínas Virais , Internalização do Vírus , Liberação de Vírus , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Nucleopoliedrovírus/metabolismo , Nucleopoliedrovírus/fisiologia , Nucleopoliedrovírus/ultraestrutura , Spodoptera/citologia , Spodoptera/metabolismo , Spodoptera/ultraestrutura , Spodoptera/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Replicação Viral , Transporte Biológico , Células Sf9
2.
Cell ; 184(21): 5419-5431.e16, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34597582

RESUMO

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.


Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Animais , Morte Celular , Sobrevivência Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Mamíferos/genética , Camundongos Endogâmicos C57BL , RNA/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
3.
Nat Struct Mol Biol ; 28(4): 388-397, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33782614

RESUMO

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.


Assuntos
Proteínas AAA/genética , Katanina/genética , Proteínas Associadas aos Microtúbulos/genética , Piridinas/química , Proteínas AAA/antagonistas & inibidores , Proteínas AAA/ultraestrutura , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Humanos , Katanina/ultraestrutura , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/genética , Microtúbulos/ultraestrutura , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Piridinas/farmacologia , Triazóis/química
5.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
6.
Sci Rep ; 9(1): 14645, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601934

RESUMO

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. They could be considered as modular building blocks assembled in distinct fashion and connected by linkers of varying lengths and sequences. Due to their intrinsic flexibility, these linkers provide proteins a subtle way to modulate interactions and explore a wide range of conformational space. In the present study, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 UIM-SH3 dual domain protein with respect to molecular recognition. We have engineered several constructs of UIM-SH3 with different length linkers or domain deletion. By means of SAXS and NMR experiments, we have shown that the modification of the linker modifies the flexibility and the dynamics of UIM-SH3. Indeed, the global tumbling of both the UIM and SH3 domain is different but not independent from each other while the length of the linker has an impact on the ps-ns time scale dynamics of the respective domains. Finally, the modification of the flexibility and dynamics of the linker has a drastic effect on the interaction of UIM-SH3 with Lys63-linked diubiquitin with a roughly eight-time weaker dissociation constant.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitinas/metabolismo , Domínios de Homologia de src , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Espalhamento a Baixo Ângulo , Ubiquitinas/ultraestrutura , Difração de Raios X
7.
Sci Adv ; 5(4): eaau7198, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30989108

RESUMO

Many cellular processes such as endosomal vesicle budding, virus budding, and cytokinesis require extensive membrane remodeling by the endosomal sorting complex required for transport III (ESCRT-III). ESCRT-III protein family members form spirals with variable diameters in vitro and in vivo inside tubular membrane structures, which need to be constricted to proceed to membrane fission. Here, we show, using high-speed atomic force microscopy and electron microscopy, that the AAA-type adenosine triphosphatase VPS4 constricts and cleaves ESCRT-III CHMP2A-CHMP3 helical filaments in vitro. Constriction starts asymmetrically and progressively decreases the diameter of CHMP2A-CHMP3 tubular structure, thereby coiling up the CHMP2A-CHMP3 filaments into dome-like end caps. Our results demonstrate that VPS4 actively constricts ESCRT-III filaments and cleaves them before their complete disassembly. We propose that the formation of ESCRT-III dome-like end caps by VPS4 within a membrane neck structure constricts the membrane to set the stage for membrane fission.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , ATPases Vacuolares Próton-Translocadoras/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Hidrólise , Microscopia de Força Atômica , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Exp Cell Res ; 372(1): 1-15, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30144444

RESUMO

Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/química , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Regulação da Expressão Gênica , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Lisossomos/ultraestrutura , Corpos Multivesiculares/ultraestrutura , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
9.
PLoS Biol ; 15(8): e2002354, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806752

RESUMO

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Micropartículas Derivadas de Células/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/sangue , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/sangue , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Éxons , Células HeLa , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/patologia , Heterozigoto , Humanos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Proteínas do Tecido Nervoso/sangue , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/sangue , Proteínas Nucleares/química , Proteínas Nucleares/genética , Tamanho da Partícula , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
10.
Nat Commun ; 8: 16064, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28714467

RESUMO

The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.


Assuntos
Adenosina Trifosfatases/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Escherichia coli , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Relação Estrutura-Atividade
11.
Elife ; 62017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28379137

RESUMO

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.


Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transporte Proteico
12.
Methods Cell Biol ; 137: 205-224, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28065306

RESUMO

The spatiotemporal characteristics of ESCRT (Endosomal Sorting Complex Required for Transport)-mediated mammalian cytokinetic abscission have been studied in recent years using quantitative high-resolution light microscopy techniques. Here we describe how to apply spinning disk live cell imaging and structured illumination microscopy (SIM) to define the dynamics and structural organization of abscission and of proteins involved in abscission in a quantitative manner. We further provide a protocol to correlate the structural data, obtained by SIM, to the dynamic information obtained by live cell recordings.


Assuntos
Citocinese/genética , Endossomos/ultraestrutura , Microscopia/métodos , Imagem Molecular/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Endossomos/genética , Células HeLa , Humanos
13.
Dev Cell ; 35(4): 397-8, 2015 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-26609952

RESUMO

In a recent issue of Cell, Chiaruttini et al. (2015) reveal the mechanical properties of the mysterious spiral filaments formed by the yeast ESCRT-III protein Snf7. The spirals are shown to be springs whose bending drives membrane deformation and perhaps membrane scission.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Bicamadas Lipídicas/química , Modelos Moleculares , Leveduras/metabolismo
14.
Cell ; 163(4): 866-79, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26522593

RESUMO

ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Bicamadas Lipídicas/química , Modelos Moleculares , Leveduras/metabolismo , Membranas Intracelulares/química , Liberação de Vírus , Leveduras/citologia
15.
Eukaryot Cell ; 14(10): 976-82, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26150415

RESUMO

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.


Assuntos
Motivos de Aminoácidos/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura
16.
J Cell Biol ; 206(6): 763-77, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25202029

RESUMO

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts.


Assuntos
Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Subunidades Proteicas/metabolismo , Animais , Cristalografia por Raios X , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Polímeros/metabolismo , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/biossíntese
17.
PLoS Pathog ; 10(4): e1004087, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24763736

RESUMO

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.


Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tombusvirus/enzimologia , Adenosina Trifosfatases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Tombusvirus/genética , Tombusvirus/ultraestrutura
18.
Mol Biol Cell ; 24(11): 1615-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23722945

RESUMO

A rich and ongoing history of cell biology research has defined the major polymer systems of the eukaryotic cytoskeleton. Recent studies have identified additional proteins that form filamentous structures in cells and can self-assemble into linear polymers when purified. This suggests that the eukaryotic cytoskeleton is an even more complex system than previously considered. In this essay, I examine the case for an expanded definition of the eukaryotic cytoskeleton and present a series of challenges for future work in this area.


Assuntos
Citoesqueleto/ultraestrutura , Drosophila melanogaster/química , Células Eucarióticas/ultraestrutura , Schizosaccharomyces/química , Actinas/química , Actinas/ultraestrutura , Animais , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/ultraestrutura , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/ultraestrutura , Citoesqueleto/química , Drosophila melanogaster/citologia , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Endossomos/química , Endossomos/ultraestrutura , Escherichia coli/química , Escherichia coli/citologia , Células Eucarióticas/química , Humanos , Multimerização Proteica , Transporte Proteico , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/ultraestrutura
19.
Mol Biol Cell ; 24(15): 2319-27, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23761076

RESUMO

The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.


Assuntos
Proteínas Arqueais/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Sulfolobus/crescimento & desenvolvimento , Proteínas Arqueais/ultraestrutura , Membrana Celular/fisiologia , Microscopia Crioeletrônica , Citocinese , Tomografia com Microscopia Eletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Sulfolobus/metabolismo , Sulfolobus/ultraestrutura
20.
Cell Microbiol ; 15(2): 213-26, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23051622

RESUMO

The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome and for the abscission step of cytokinesis. ESCRT-III proteins form polymers that constrict membrane tubes, leading to fission. We have used electron cryomicroscopy to determine the molecular organization of pleiomorphic ESCRT-III CHMP2A-CHMP3 polymers. The three-dimensional reconstruction at 22 Å resolution reveals a helical organization of filaments of CHMP molecules organized in a head-to-tail fashion. Protease susceptibility experiments indicate that polymerization is achieved via conformational changes that increase the protomer stability. Combinatorial siRNA knockdown experiments indicate that CHMP3 contributes synergistically to HIV-1 budding, and the CHMP3 contribution is ~ 10-fold more pronounced in concert with CHMP2A than with CHMP2B. This is consistent with surface plasmon resonance affinity measurements that suggest sequential CHMP4B-CHMP3-CHMP2A recruitment while showing that both CHMP2A and CHMP2B interact with CHMP4B, in agreement with their redundant functions in HIV-1 budding. Our data thus indicate that the CHMP2A-CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , HIV-1/química , Liberação de Vírus/fisiologia , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , HIV-1/fisiologia , Modelos Moleculares , Peptídeo Hidrolases/química , Polimerização , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteólise , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Ressonância de Plasmônio de Superfície
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